CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/272,931, filed Nov. 19, 2009, herein incorporated by reference in its entirety.
FIELD OF THE INVENTION The present invention relates to the use of a biosynthetic system and proteins for preparing a vaccine. In addition, the invention relates to a recombinant prokaryotic biosynthetic system having an epimerase that initiates the synthesis of an oligo- or polysaccharide with a specified monosaccharide at the reducing terminus. The invention further relates to N-glycosylated proteins produced with glycans in an expression system and bioconjugate vaccines made from said N-glycosylated proteins comprising immunogenic glycans, and provides methods for producing N-glycosylated proteins.
BACKGROUND OF THE INVENTION Glycoproteins are proteins that have one or more covalently attached sugar polymers. N-linked protein glycosylation is an essential and conserved process occurring in the endoplasmic reticulum of eukaryotic organisms. It is important for protein folding, oligomerization, stability, quality control, sorting and transport of secretory and membrane proteins (Helenius. A., and Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019-1049).
Protein glycosylation has a profound influence on the immunogenicity, the stability and the half-life of a protein. In addition, glycosylation can assist the purification of proteins by chromatography, e.g. affinity chromatography with lectin ligands bound to a solid phase interacting with glycosylated moieties of the protein. It is therefore established practice to produce many glycosylated proteins recombinantly in eukaryotic cells to provide biologically and pharmaceutically useful glycosylation patterns.
WO 200307467 (Aebi et al.) demonstrated that the food-borne pathogen Campylobacter jejuni, which is a bacterium, could N-glycosylate its proteins, which was a unique feature among known prokaryotic organisms except for certain species of archaea. The machinery required for glycosylation is encoded by 12 genes that are clustered in the so-called pgl locus. Disruption of N-glycosylation affects invasion and pathogenesis of C. jejuni but is not lethal as in most eukaryotic organisms (Burda P. and M. Aebi, (1999). The dolichol pathway of N-linked glycosylation. Biochem Biophys Acta 1426(2):239-57). It is possible to reconstitute the N-glycosylation of C. jejuni proteins by recombinantly expressing the pgl locus and acceptor glycoprotein in E. coli the same time (Wacker et al. (2002). N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790-1793).
N-glycans have a glycan attached to a consensus sequence in a protein. The known N-glycosylation consensus sequence in a protein allows for the N-glycosylation of recombinant target proteins in prokaryotic organisms. Such organisms comprise an oligosaccharyl transferase (“OT”; “OTase”), such as, for example, an oligosaccharyl transferase of C. jejuni, which is an enzyme that transfers the glycan to the consensus sequence of the protein.
WO 200307467 (Aebi et al.) teaches a prokaryotic organism into which is introduced a nucleic acid encoding for (i) specific glycosyltransferases for the assembly of an oligosaccharide on a lipid carrier, (ii) a recombinant target protein comprising a consensus sequence “N—X—S/T”, wherein X can be any amino acid except proline, and (iii) an oligosaccharyl transferase, such as, for example, an oligosaccharyl transferase of C. jejuni that covalently links said oligosaccharide to the consensus sequence of the target protein. Said prokaryotic Organism produces N-glycans with a specific structure which is defined by the type of the specific glycosyltransferases.
WO 2006/119987 (Aebi et al.) describes proteins, as well as means and methods for producing proteins, with efficiency for N-glycosylation in prokaryotic organisms in vivo. It further describes an efficient introduction of N-glycans into recombinant proteins for modifying immunogenicity, stability, biological, prophylactic and/or therapeutic activity of said proteins, and the provision of a host cell that efficiently displays recombinant N-glycosylated proteins of the present invention on its surface. In addition, it describes a recombinant N-glycosylated protein comprising one or more of the following N-glycosylated optimized amino acid sequence(s):
D/E-X-N-Z-S/T (optimized consensus sequence),
wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated partial amino acid sequence(s) is introduced. The introduction of specific partial amino acid sequence(s) (optimized consensus sequence(s)) into proteins leads to proteins that are efficiently N-glycosylated by an oligosaccharyl transferase in these introduced positions.
The biosynthesis of different polysaccharides is conserved in bacterial cells. The polysaccharides are assembled on carrier lipids from common precursors (activated sugar nucleotides) at the cytoplasmic membrane by different glycosyltransferases with defined specificity. Lipopolysaccharides (“LPS”) are provided in gram-negative bacteria only, e.g. Shigella spp., Pseudomonas spp. and E. coli (ExPEC, EHEC).
The synthesis of LPS starts with the addition of a monosaccharide to the carrier lipid undecaprenyl phosphate (“Und-P-P”) at the cytoplasmic side of the membrane. The antigen is built up by sequential addition of monosaccharides from activated sugar nucleotides by different glycosyltransferases, and the lipid-linked polysaccharide is flipped through the membrane by a flippase. The antigen-repeating unit is polymerized by an enzymatic reaction. The polysaccharide is then transferred to the Lipid A by the Ligase WaaL forming the LPS that is exported to the surface, whereas the capsular polysaccharide is released from the carrier lipid after polymerization and exported to the surface. The biosynthetic pathway of these polysaccharides enables the production of LPS bioconjugates in vivo, capturing the polysaccharides in the periplasm to a protein carrier.
Such synthesized complexes of oligo- or polysaccharides (i.e., sugar residues) and proteins (i.e., protein carriers) can be used as conjugate vaccines to protect against a number of bacterial infections. Conjugate vaccines have been successfully used to protect against bacterial infections. The conjugation of an antigenic polysaccharide to a protein carrier is required for protective memory response, as polysaccharides are T-cell independent immunogens. Polysaccharides have been conjugated to protein carriers by different chemical methods, using activation reactive groups in the polysaccharide as well as the protein carrier.
Conjugate vaccines can be administered to children to protect against bacterial infections and also can provide a long lasting immune response to adults. Constructs of WO 2009104074 (Fernandez, et al.) have been found to generate an IgG response in animals. It has been found that an IgG response to a Shigella O-specific polysaccharide-protein conjugate vaccine in humans correlates with immune protection in humans. (Passwell, J. H. et al., “Safety and Immunogenicity of Improved Shigella O-Specific Polysaccharide-Protein Conjugate Vaccines in Adults in Israel” Infection and Immunity, 69(3):1351-1357 (March 2001).) It is believed that the polysaccharide (i.e. sugar residues) triggers a short-term immune response that is sugar-specific. Indeed, the human immune system generates a strong response to specific polysaccharide surface structures of bacteria, such as O-antigens and capsular polysaccharides. However, since the immune response to polysaccharides is IgM dependent, the immune system develops no memory. The protein carrier that carries the polysaccharide triggers an IgG response that is T-cell dependent and that provides long lasting protection since the immune system develops memory.
E. coli O157 is an enterohemorrhagic strain responsible for approximately two-thirds of all recent cases of hemolytic-uremic syndrome and poses serious human health concerns (Law, D. (2000) J. App. Microbiol., 88, 729-745; Wang, L., and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551).
Escherichia coli strain O157 produces an O-antigen containing the repeating tetrasaccharide unit (4-N-acetyl perosamine→fucose→glucose→GalNAc) (α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc) (Perry, M. B., MacLean, L. and Griffith, D. W. (1986) Biochem. Cell. Biol., 64, 21-28). The tetrasaccharide is preassembled on undecaprenyl pyrophosphate. The E. coli cell envelope contains an inner plasma membrane, a stress-hearing peptidoglycan layer and an asymmetric outer membrane consisting of a phospholipid inner monolayer and an outer monolayer composed of bacterial LPS. LPS contains three components, the lipid A anchor, the 3-deoxy-D-manno-oct-2-ulosonic acid-containing core, and the O-antigen region (see: Raetz, C. R. H. and Whitfield, C. (2002) Annu. Rev. Biochem., 71, 635-700; Whitfield, C. (2006) Ann. Rev. Biochem. 75, 39-68; Samuel, G. and Reeves, P. R. (2003) Carbohydrate Research, 338, 2503-2519; and refs, therein for reviews on the assembly of O-antigens of bacterial LPS).
The O-antigen components of bacterial LPS are large, extremely diverse polysaccharides that can be either homopolymeric, composed of a single repeating monosaccharide, or heteropolymeric, containing 10-30 repeats of 3-6 sugar units (Reeves, P. R., Hobbs, M., Valvano, M. A., Skurnik, M., Whitfield, C., Coplin, D., Kido, N., Klena, J., Maskell, D., Raetz, C. R. H., and Rick, P. D. (1996) Trends Microbial., 4, 495-503). O-Antigens are, Thus, the Dominant Feature of the bacterial cell surface and constitute important determinants of virulence and pathogenicity (Law, D. (2000) J. App. Microbiol., 88, 729-745; Spears, K. J., Roe, A. J. and Golly, D. L. (2006) FEMS Microbiol. Lett., 255, 187-202; Liu, B., Knirel, Y. A., Feng, L., Perepelov, A. V., Senchenkova, S. N., Wang, Q., Reeves, P. R. and Wang, L (2008) FEMS Microbiol. Rev. 32, 627-653; Stenutz, R., Weintraub, A. and Widmalm, G. (2006) FEMS Microbiol. Rev. 30, 382-403). E. coli strains with more than 180 individual O-serotypes, attributed to unique O-antigen structures, have been identified (Stenutz, R., Weintraub, A. and Widmalm, G. (2006) FEMS Microbiol. Rev. 30, 382-403).
O-antigen repeat units are pre-assembled on the cytosolic face of the inner membrane attached to undecaprenyl pyrophosphate. The lipid-linked repeat units diffuse transversely (flip-flop) to the periplasmic surface of the inner membrane and are polymerized before transport to the outer membrane and ligation to LPS. Most heteropolymeric O-antigen repeat units have either N-acetylglucosamine (“GlcNAc”) or N-acetylgalactosamine (“GalNAc”) at the reducing terminus.
It had been assumed that the biosynthesis of the lipid intermediates is initiated by the transfer of GlcNAc-9 or GalNAc-P from their respective sugar nucleotide derivatives to undecaprenyl monophosphate (“Und-P”) catalyzed by WecA (Samuel, G. and Reeves, P. R. (2003) Carbohydrate Research, 338, 2503-2519; Alexander, D, C. and Valvano, M. A. (1994) J. Bacteriol., 176, 7079-7084; Zhang, L., Radziejewska-Lebrecht, J., Krajewska-Pietrasik, D., Tolvanen, P. and Skurkik. M. (1997) Mol. Microbiol. 23, 63-76; Amor, P. A. and Whitfield, C. (1997) Mol. Microbiol. 26 (145-161); Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). Although the properties and specificity of the GlcNAc-phosphotransferase activity of WecA have been characterized (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322), the conclusion that WecA catalyzes the synthesis of GalNAc-P-P-Und was based on genetic studies (Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). Such earlier genetic studies indicated that the biosynthesis of the lipid-linked tetrasaccharide intermediate was initiated by the enzymatic transfer of GalNAc-P from UDP-GalNAc to Und-P catalyzed by WecA (Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). However, there was no direct enzymological evidence demonstrating that WecA utilizes UDP-GalNAc as a GalNAc-P donor.
Furthermore, the E. coli O55 gne and gne1 genes were previously proposed to encode a UDP-GlcNAc 4-epimerase (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625; Guo, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609). Previous reports identified two genes from E. coli O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) and E. coli O86 (Guo, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609), E. coli O55 gne and E. coli O86 gne1, respectively, that are 100% identical to a Z3206 gene within the same gene family.
Accordingly, one of skill would have been led to believe that the Z3206 gene also encodes a UDP-GlcNAc/UDP-GalNAc epimerase.
BRIEF SUMMARY OF THE INVENTION It has now been surprisingly discovered that an epimerase encoded by the 3206 gene in E. coli O157 catalyzes a reaction that synthesizes N-acetylgalactosamine (“GalNAc”) undecaprenyl pyrophosphate, which initiates the formation of an oligo- or polysaccharide.
In one aspect, the present invention relates to a recombinant prokaryotic biosynthetic system that produces all or a portion of a polysaccharide comprising an epimerase that synthesizes GalNAc on undecaprenyl pyrophosphate. The invention further includes glycosyltransferases that synthesize all or a portion of a polysaccharide having GalNAc at the reducing terminus, and still further includes glycosyltransferases that synthesize all or a portion of an antigenic polysaccharide having GalNAc at the reducing terminus.
In another aspect, the invention is directed to an epimerase to produce GalNAc on undecaprenyl pyrophosphate, and, in a further aspect, the epimerase is encoded by the Z3206 gene.
In an additional aspect, the present invention is directed to an expression system for producing an N-glycosylated protein comprising: a nucleotide sequence encoding an oligosaccharyl transferase; a nucleotide sequence encoding a protein carrier; at least one oligo- or polysaccharide gene cluster from at least one bacterium, wherein the polysaccharide contains GalNAc at the reducing terminus; and a nucleic acid sequence encoding an epimerase.
In a still further aspect, the instant invention is directed to a recombinant prokaryotic biosynthetic system comprising Z3206 gene which encodes an epimerase that converts GlcNAc-P-P-Und to GalNAc-P-P-Und.
In yet an additional aspect, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising E. coli O55 gne gene or E. coli O86 gne1 gene which encodes an epimerase that converts GlcNAc-P-P-Und to GalNAc-P-P-Und.
In yet another aspect, the present invention relates to an N-glycosylated protein comprising at least one introduced consensus sequence, D/E-X-N-Z-S/T, wherein X and Z can be any natural amino acid except proline, and a glycan having N-acetylgalactosamine at the reducing terminus.
In still another aspect, the present invention is directed to a bioconjugate vaccine comprising an N-glycosylated protein having at least one introduced consensus sequence, D/E-X-N-Z-S/T, wherein X and Z can be any natural amino acid except proline: an immunogenic glycan having N-acetylgalactosamine at the reducing terminus; and an adjuvant.
In an addition aspect, the invention relates to method for producing an N-linked glycosylated protein in a host cell comprising nucleic acids encoding: glycosyltransferases that assemble at least one oligo- or polysaccharide from at least one bacterium containing GalNAc at the reducing terminus; a protein carrier; an oligosaccharyl transferase; and an epimerase.
In a further aspect, the present invention relates to the use of a biosynthetic system and proteins for preparing a bioconjugate vaccine.
In an additional aspect, the present invention is directed to methods for producing mono-, oligo- and polysaccharides, and in a still further aspect the invention directed to methods for producing antigenic glycans and N-glycosylated proteins.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the time course of [3H]GlcNAc/GalNAc-P-P-Und synthesis by membrane fractions from E. coli O157. The membrane fraction from E. coli strain O157 was incubated with UDP-[3H]GlcNAc for the indicated times at 37° C. The [3H]lipid products were extracted and the incorporation of [3H]GlcNAc into [3H]GlcNAc-P-P-Und (O) and [3H]GalNAc-P-P-Und (•) was assayed as described in Example 2.
FIG. 2 shows the proposed biosynthetic pathway for the formation of GalNAc-P-P-Und from GlcNAc-P-P-Und.
FIGS. 3A, 3B, 3C, and 3D shows purification and characterization of [3H]GalNAc-P-P-Und synthesized by membrane fractions from E. coli strain O157. Membrane fractions from E. coli O157 were incubated with UDP-[3H]GlcNAc, and the [3H]GalNAc lipids were purified as described in Example 3. FIG. 3A, preparative thin layer chromatogram of [3H]HexNAc lipids on borate-impregnated silica gel G (Quantum 1) after purification on DEAE-cellulose is shown. FIG. 3B, thin layer chromatography of purified [3H]GalNAc-P-P-Und on borate-impregnated silica gel G (Baker, Si250) after recovery from the preparative plate in panel A is shown. FIG. 3C. descending paper chromatogram (borate-impregnated Whatman No. 1 paper) of the [3H]-amino sugar recovered after mild acid hydrolysis of [3H]GalNAc-P-P-Und purified in FIG. 3B is shown. FIG. 3D, descending paper chromatogram (Whatman No. 3MM) of the [3H]HexNAc-alditol produced by reduction of the [3H] amino sugar from FIG. 3C with NaBH4.
FIGS. 4A and 4B shows metabolic labeling of E. coli 21546 cells and E. coli 21546 cells after transformation with pMLBAD:Z3206. E. coli 21546 (FIG. 4A) and E. coli 21546:pMLBAD/Z3206 (FIG. 4B) were labeled metabolically with [3H]GlcNAc for 5 min at 37° C. [3H]GlcNAc/GalNAc-P-P-Und were extracted, freed of water soluble contaminants and separated by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) as described in Example 3. Radioactive lipids were detected using a Bioscan chromatoscanner. The chromatographic positions of GalNAc-P-P-Und and GlcNAc-P-P-Und are indicated by arrows.
FIGS. 5A, 5B, 5C, and 5D shows thin layer chromatography of [3H]GlcNAc/GalNAc-P-P-Und formed by incubation of membrane fractions from E. coli strains with UDP-[3H]GlcNAc. Membrane fractions from E. coli strains K12 (FIG. 5A), O157 (FIG. 5B), 21546 (FIG. 5C), and 21546:pMLBAD/Z3206 (FIG. 5D) were incubated with UDP-[3H]GlcNAc for 10 min at 37° C., and the [3H]lipid products were extracted, freed of water-soluble contaminants by partitioning, and separated by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) as described in Example 3. The chromatographic positions of GalNAc-P-P-Und and GlcNAc-P-P-Und are indicated by arrows.
FIGS. 6A, 6B, and 6C shows discharge of GlcNAc-P by incubation with UMP. Membrane fractions from E. coli 21546:Z3206 were preincubated with UDP-[3H]GlcNAc to enzymatically label GlcNAc-P-P-Und for 10 min (FIG. 6A) at 37° C. followed by a second incubation period with 1 mM UMP included for either 1 min (FIG. 6B) or 2 min (FIG. 6C). After the indicated incubation periods [3H]GlcNAc/GalNAc-P-P-Und were extracted and resolved by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) as described in Example 3. The chromatographic positions of GalNAc-P-P-Und and GlcNAc-P-P-Und are indicated by arrows.
FIGS. 7A, 7B, 7C, 7D, 7E, and 7F shows conversion of exogenous [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und to the pertinent [3H]HexNAc-P-P-Und product catalyzed by membranes from strain 21546 expressing Z3206. Membrane fractions from E. coli strain 21546 (FIG. 7B and FIG. 7E) and 215461:pMLBAD/Z3206 (FIG. 7C and FIG. 7F) were incubated with purified [3H]GlcNAc-P-P-Und (FIG. 7A, FIG. 7B, and FIG. 7C) or [3H]GalNAc-P-P-Und (panels at FIG. 7D, FIG. 7E, and FIG. 7F) (dispersed ultrasonically in 1% Triton X-100) for 1 min at 37° C. [3H]GlcNAc/GalNAc-P-P-Und were extracted, resolved by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) and detected with a Bioscan AR2000 radiochromatoscanner as described in Example 3.
FIG. 8 shows SDS-PAGE analysis of unglycosylated and glycosylated AcrA protein. Periplasmic extracts prepared from E. coli DH5α cells carrying the AcrA expression plasmid and the pgl operon Agile complemented with pMLBAD:Z3206 (lane 1), pMLBAD:gne (lane 2) or the vector control pMLBAD (lane 3) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. AcrA and its glycosylated forms were detected with anti AcrA antisera. The position of bands corresponding to unglycosylated (AcrA) and glycosylated AcrA (gAcrA) is indicated.
FIG. 9 shows the genes that have been identified by Liu B et al. (Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27).
FIG. 10 is a scheme showing the DNA region containing the genes required to synthesize the S. flexneri 6 O antigen.
FIG. 11 shows expression of the S. flexneri 6 O antigen in E. coli. LPS was visualized by either silver staining or by transfer to nitrocellulose membranes and detection by antibodies directed against S. flexneri 6.
FIG. 12 shows HPLC of O antigen. LLO analysis of E. coli cells (SCM3) containing S. flexneri—Z3206, E. coli cells (SCM3) containing S. flexneri+Z3206 or empty E. coli (SCM3) cells.
FIG. 13 shows Western blot of Nickel purified protein, E. coli cells expressing EPA, pglB and S. flexneri 6 O-antigen+/−Z3206
DETAILED DESCRIPTION OF THE INVENTION The present invention encompasses a recombinant prokaryotic biosynthetic system comprising nucleic acids encoding an epimerase that synthesizes an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus, and N-glycosylated proteins having N-acetylgalactosamine at the reducing terminus of the glycan.
The term “partial amino acid sequence(s)” is also referred to as “optimized consensus sequence(s)” or “consensus sequence(s).” The optimized consensus sequence is N-glycosylated by an oligosaccharyl transferase (“OST,” “OTase”), much more efficiently than the regular consensus sequence “N—X-ST.”
In general, the term “recombinant N-glycosylated protein” refers to any poly- or oligopeptide produced in a host cell that does not naturally comprise the nucleic acid encoding said protein. In the context of the present invention, this term refers to a protein produced recombinantly in a prokaryotic host cell, for example, Escherichia spp., Campylobacter spp., Salmonella spp., Shigella spp., Helicobacter spp., Pseudomonas spp., Bacillus spp., and in further embodiments Escherichia cell, Campylobacter jejuni, Salmonella typhimurium etc., wherein the nucleic acid encoding said protein has been introduced into said host cell and wherein the encoded protein is N-glycosylated by the OTase, said transferase enzyme naturally occurring in or being introduced recombinantly into said host cell.
In accordance with the internationally accepted one letter code for amino acids the abbreviations D, E, N, S and T denote aspartic acid, glutamic acid, asparagine, serine, and threonine, respectively.
Proteins according to the invention comprise one or more of an optimized consensus sequence(s) D/E-X-N-Z-S/T that is/are introduced into the protein and N-glycosylated. Hence, the proteins of the present invention differ from the naturally occurring C. jejuni N-glycoproteins which also contain the optimized consensus sequence but do not comprise any additional (introduced) optimized consensus sequences.
The introduction of the optimized consensus sequence can be accomplished by the addition, deletion and/or substitution of one or more amino acids. The addition, deletion and/or substitution of one or more amino acids for the purpose of introducing the optimized consensus sequence can be accomplished by chemical synthetic Strategies, which, in view of the instant invention, would be well known to those skilled in the art such as solid phase-assisted chemical peptide synthesis. Alternatively, and preferred for larger polypeptides, the proteins of the present invention can be prepared by recombinant techniques that would be art-standard techniques in light of the invention.
The proteins of the present invention have the advantage that they may be produced with high efficiency and in any host. In one embodiment of the invention, the host comprises a functional pgl operon from Campylobacter spp., for example, from C. jejuni. In further embodiments, oligosaccharyl transferases from Campylobacter spp. for practicing the invention are from Campylobacter coli or Campylobacter lari. In view of the invention, oligosaccharyl transferases would be apparent to one of skill in the art. For example, oligosaccharyl transferases are disclosed in references such as Szymanski, C. M. and Wren, B. W. (2005) Protein glycosylation in bacterial mucosal pathogens, Nat. Rev. Microbiol. 3:225-237. The functional pgl operon may be present naturally when said prokaryotic host is Campylobacter spp., or, for example, C. jejuni. However, as demonstrated before in the art and mentioned above, the pgl operon can be transferred into cells and remain functional in said new cellular environment.
The term “functional pgl operon from Campylobacter spp., preferably C. jejuni” is meant to refer to the cluster of nucleic acids encoding the functional oligosaccharyl transferase (OTase) of Campylobacter spp., for example, C. jejuni, and one or more specific glycosyltransferases capable of assembling an oligosaccharide on a lipid carrier, and wherein said oligosaccharide can be transferred from the lipid carrier to the target protein having one or more optimized amino acid sequence(s): D/E-X-N-Z-S/T by the OTase. It to be understood that the term “functional pgl operon from Campylobacter spp., preferably C. jejuni” in the context of this invention does not necessarily refer to an operon as a singular transcriptional unit. The term merely requires the presence of the functional components for N-glycosylation of the recombinant protein in one host cell. These components may be transcribed as one or more separate mRNAs and may be regulated together or separately. For example, the term also encompasses functional components positioned in genomic DNA and plasmid(s) in one host cell. For the purpose of efficiency, in one embodiment all components of the functional pgl operon are regulated and expressed simultaneously.
The oligosaccharyl transferase can originate, in some embodiments, from Campylobacter spp., and in other embodiments, from C. jejuni. In additional embodiments, the oligosaccharyl transferase can originate from other organisms which are known to those of skill in the art as having an oligosaccharyl transferase, such as, for example, Wolinella spp. and eukaryotic organisms.
The one or more specific glycosyltransferases capable of assembling an oligosaccharide on a lipid carrier may originate from the host cell or be introduced recombinantly into said host cell, the only functional limitation being that the oligosaccharide assembled by said glycosyltransferases can be transferred from the lipid carrier to the target protein having one or more optimized consensus sequences by the OTase. Hence, the selection of the host cell comprising specific glycosyltransferases naturally and/or replacing specific glycosyltransferases naturally present in said host as well as the introduction of heterologous specific glycosyltransferases will enable those skilled in the art to vary the N-glycans bound to the optimized N-glycosylation consensus site in the proteins of the present invention.
As a result of the above, the present invention provides for the individual design of N-glycan-patterns on the proteins of the present invention. The proteins can therefore be individualized in their N-glycan pattern to suit biological, pharmaceutical and purification needs.
In embodiments of the present invention, the proteins may comprise one but also more than one, such as at least two, at least 3 or at least 5 of said N-glycosylated optimized amino acid sequences.
The presence of one or more N-glycosylated optimized amino acid sequence(s) in the proteins of the present invention can be of advantage for increasing their immunogenicity, increasing their stability, affecting their biological activity, prolonging their biological half-life and/or simplifying their purification.
The optimized consensus sequence may include any amino acid except proline in position(s) X and Z. The term “any amino acids” is meant to encompass common and rare natural amino acids as well as synthetic amino acid derivatives and analogs that will still allow the optimized consensus sequence to be N-glycosylated by the OTase. Naturally occurring common and rare amino acids are preferred for X and Z. X and Z may be the same or different.
It is noted that X and Z may differ for each optimized consensus sequence in a protein according to the present invention.
The N-glycan hound to the optimized consensus sequence will be determined by the specific glycosyltransferases and their interaction when assembling the oligosaccharide on a lipid carrier for transfer by the OTase. In view of the instant invention, those skilled in the art would be able to design the N-glycan by varying the type(s) and amount of the specific glycosyltransferases present in the desired host cell.
“Monosaccharide” as used herein refers to one sugar residue. “Oligo- and polysaccharide” refer to two or more sugar residues. The term “glycans” as used herein refers to mono-, oligo- or polysaccharides. “N-glycans” are defined herein as mono-, oligo- or polysaccharides of variable compositions that are linked to an ε-amide nitrogen of an asparagine residue in a protein via an N-glycosidic linkage. In an embodiment, the N-glycans transferred by the OTase are assembled on an undecaprenol pyrophosphate (“Und-P-P”) lipid-anchor that is present in the cytoplasmic membrane of gram-negative or positive bacteria. They are involved in the synthesis of O antigen, O polysaccharide and peptidoglycan (Bugg, T. D., and Brandish, P. E. (1994). From peptidoglycan to glycoproteins: common features of lipid-linked oligosaccharide biosynthesis. FEMS Microbiol Lett 119, 255-262; Valvano, M. A. (2003). Export of O-specific lipopolysaccharide. Front Biosci 8, s452-471).
Studies were conducted to determine whether the biosynthesis of a lipid-linked repeating tetrasaccharide (4-N-acetyl perosamine→fucose→glucose→GalNAc) was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, 0157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[3H]GalNAc, neither the enzymatic synthesis of [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[3H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und, confirming that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of an epimerase was also confirmed by showing that exogenous [3H]GlcNAc-P-P-Und was converted to [3H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [3H]GlcNAc, both [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [3H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [3H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, it was confirmed that GalNAc-P-P-Und is synthesized reversibly by a GlcNAc-P-P-Und epimerase following the formation of GlcNAc-P-P-Und by WecA in E. coli O157.
The initiating reaction of E. coli O157 O-antigen subunit assembly was investigated to confirm that GalNAc-P-P-Und synthesis is catalyzed by some previously unknown mechanism rather than by WecA. The evidence presented herein shows that GalNAc-P-P-Und is not synthesized by GalNAc-P transfer from UDP-GalNAc catalyzed by WecA but rather by the reversible epimerization of the 4-OH of GlcNAc-P-P-Und catalyzed by an epimerase encoded by the Z3206 gene in E. coli O157.
Accordingly, the invention encompasses a novel biosynthetic pathway for the assembly of an important bacterial cell surface component as well as a new biosynthetic route for the synthesis of GalNAc-P-P-Und. A further embodiment of the invention includes the bacterial epimerase as a new target for antimicrobial agents.
E. coli O157 synthesizes an O-antigen with the repeating tetrasaccharide structure (4-N-acetyl perosamine→fucose→glucose→GalNAc). It is shown herein that the biosynthesis of the lipid-linked tetrasaccharide intermediate was not initiated by the enzymatic transfer of GalNAc-P from UDP-GalNAc to Und-P catalyzed by WecA, contrary to earlier genetic studies (Wang. L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). The invention described herein, obtained by homology searches and then confirmed by results from genetic, enzymology, and metabolic labeling experiments, demonstrates that WecA does not utilize UDP-GalNAc as a substrate, but that WecA is required to synthesize GlcNAc-P-P-Und which is then reversibly converted to GalNAc-P-P-Und by an epimerase encoded by the Z3206 gene in strain O157.
The Z3206 gene of the present invention belongs to a family of genes present in several strains that produce surface O-antigen repeat units containing GalNAc residues at their reducing termini (Table 1). The Z3206 gene sequence is shown in SEQ ID NO: 1. Previous reports identified two genes from E. coli O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) and E. coli O86 (Gun, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Comm., 356, 604-609), E. coli O55 gne and E. coli O86 gne1, respectively, that are 100% identical to a Z3206 gene (Table 1). The E. coli O55 gne gene sequence is shown as SEQ ID NO: 3, and E. coli O86 gne1 gene sequence is shown as SEQ ID NO: 5.
TABLE 1
Correlation of Z3206 gene in bacterial strains expressing O-antigen
chains with GalNAc at the reducing termini.
GalNAc
% Identity at the reducing
with terminus of O-antigen
Z3206 repeat unit
E. coli O55 gne (SEQ ID NO: 3) 100 Yes
E. coli O86 gnel (SEQ ID NO: 5) 100 Yes
Shigella boydii O18 gne (SEQ ID 88 Yes
NO: 7)
Salmonella enterica O30 gne 94 Yes
(SEQ ID NO: 9)
C. jejuni gne (SEQ ID NO: 11) 21 No
E. coli K12 galE (SEQ ID NO: 13) 27 No
E. coli O86 gne2 (SEQ ID NO: 15) 18 Yes
Accordingly, we conclude that E. coli O55 gne and E. coli O86 gne1 also encode epimerases capable of converting GlcNAc-P-P-Und to GalNAc-P-P-Und in strains O55 and O86, respectively, which also produce O-antigen repeat units with GalNAc at the reducing termini (Table 1).
Two experimental approaches in this study indicate that the Z3206 protein does not catalyze the epimerization of UDP-GlcNAc to UDP-GalNAc in strain O157. First, when membranes from strain O157 were incubated with [3H]UDP-GalNAc, neither [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected (Table 3). If Z3206 catalyzed the conversion of [3H]UDP-GalNAc to [3H]UDP-GlcNAc, it would be expected that [3H]GlcNAc-P-P-Und should be observed. Second, we have shown that hemagglutinin-tagged Z3206 was incapable of complementing the UDP-GalNAc-dependent C. jejuni N-glycosylation reporter system (FIG. 8).
E. coli O55 gne gene from strain O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) was also assayed for epimerase activity by incubating crude extracts with UDP-GalNAc and indirectly assaying the conversion to UDP-GlcNAc by measuring an increase in reactivity with p-dimethylaminobenzaldehyde after acid hydrolysis. In both studies, the formation of the product was based on changes in reactivity with p-dimethylaminobenzaldehyde, and not a definitive characterization of the sugar nucleotide end product. A 90% pure polyhistidine-tagged E. coli O86 gne1 was also shown to have a low level of UDP-glucose epimerase activity relative to Gne2 in a coupled assay.
Accordingly, an embodiment of the invention is directed to a recombinant prokaryotic biosynthetic system containing Z3206 gene, E. coli O55 gne gene or E. coli O86 gne1 gene that converts GlcNAc-P-P-Und to GalNAc-P-P-Und.
It is significant that E. coli O86, which synthesizes an O-antigen containing two GalNAc residues, which would presumably require UDP-GalNAc as the glycosyl donor for the additional, non-reducing terminal GalNAc, also possesses an additional GlcNAc 4-epimerase gene, termed gne2, within the O-antigen gene cluster (Guo. B, Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609). This additional epimerase gene has high homology with the galE gene of the colanic acid gene cluster and appears to be a UDP-GlcNAc 4-epimerase capable of synthesizing UDP-GalNAc.
The Z3206 gene appears to be highly conserved in E. coli O-serotypes initiated with GalNAc. In a recent study, 62 E. coli strains, with established O-antigen repeat unit structures, were screened for expression of Z3206 by a polymerase chain reaction based method using nucleotide primers designed to specifically detect the E. coli O157 Z3206 gene (Wang, L., Huskic, Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625). In this study Z3206 was detected in 16 of the 22 E. coli strains that were known to contain GalNAc, and in only 4 of the 40 strains lacking GalNAc. Moreover, a similar screen of the 22 GalNAc-containing strains with primers designed to detect an alternative epimerase with UDP-GlcNAc 4-epimerase activity (the GalE gene of E. coli O113) detected no strains carrying this gene, indicating that Z3206 is the GlcNAc 4-epimerase gene most commonly associated with the presence of a reducing-terminal GalNAc in O-antigen repeat units of E. coli.
Analysis of the Z3206 protein sequence by a variety of web-based topological prediction algorithms indicates that the Z3206 protein is not highly hydrophobic. The majority of the topological prediction algorithms indicate that Z3206 is a soluble 37 kDa protein, although TMPred (Hofmann, K., and Stoffel, W. (1993) Biol. Chem. Hoppe-Seyler 374, 166 (abstr.)) predicted a single weak N-terminal transmembrane helix. However, Western blotting after SDS-PAGE of cellular fractions from E. coli cells expressing hemagglutinin-tagged Z3206 clearly shows that the tagged protein is associated with the particulate fraction following hypotonic lysis of the cells. Preliminary experiments show that the protein remains associated with the particulate fraction following incubation of the membrane fraction with 1 M KCl, but is solubilized in an active form by incubation with 0.1% Triton X-100.
E. coli O157 Z3206 has significant sequence homology with the short-chain dehydrogenase/reductase family of oxido-reductases including the GXXGXXG motif (Rossman fold), consistent with the NAD(P) binding pocket (Allard, S. T. M., Giraud, M. F., and Naismith, J. H. (2001) Cell. Mol. Life Sci. 58, 1650-1655) and the conserved SX24YX3K sequence, involved in proton abstraction and donation (Field, R. A. and Naismith, J. H. (2003) Biochemistry 42, 7637-7647). Molecular modeling based on crystal structures of UDP-Glc 4-epimerase, another member of the short-chain dehydrogenase/reductase family, suggests that, after hydride abstraction, the 4-keto intermediate rotates around the β phosphate of UDP to present the opposite face of the keto intermediate and allow re-insertion of hydride from the opposite side, thus inverting the configuration of the hydroxyl at carbon 4. The presence of these conserved sequences suggests that Z3206 likely functions via a similar mechanism. Although the equilibrium distribution of the epimerase products, seen in FIG. 7, seems to favor the formation of GlcNAc-P-P-Und, the utilization of GalNAc-P-P-Und for O-antigen repeat unit assembly would drive the epimerization reaction in the direction of GalNAc-P-P-Und by mass action.
Epimerization of the glycosyl moieties of polyisoprenoid lipid intermediates has not been widely reported in nature. In one previous study the 2-epimerization of ribosyl-P-decaprenol to form arabinosyl-P-decaprenol, an arabinosyl donor in arabinogalactan biosynthesis in mycobacteria, was reported (Mikusová, K., Huang, H., Yagi, T., Holsters, M., Vereecke, D., D'Haeze, W., Scherman, M. S., Brennan, P. J., McNeil, M. R., and Crick, D. C. (2005) J. Bacterial. 187, 8020-8025). Arabinosyl-P-decaprenol is formed via a two-step oxidation/reduction reaction requiring two mycobacterial proteins, Rv3790 and Rv3791. Although epimerization was modestly stimulated by the addition of NAD and NADP, neither Rv3790 nor Rv3791 contain either the Rossman fold or the SX24YXXXK motif, characteristic of the short-chain dehydrogenase/reductase family (Allard, S. T. M., Giraud, M.-F. and Naismith, J. H. (2001) Cell. Mal. Life Sci. 58, 1650-1655; Field, R. A. and Naismith, J. H. (2003) Biochemistry 42, 7637-7647).
In summary, a novel biosynthetic pathway for the formation of GalNAc-P-P-Und by the epimerization of GlcNAc-P-P-Und, is described.
Several antibiotics have been shown to inhibit the synthesis of GlcNAc-P-P-Und, but are limited in their utility because they also block the synthesis of GlcNAc-P-P-dolichol, the initiating dolichol-linked intermediate of the protein N-glycosylation pathway. Although GlcNAc-P-P-dolichol is a structurally related mammalian counterpart of the bacterial glycolipid intermediate, GlcNAc-P-P-Und, there is no evidence for a similar epimerization reaction converting GlcNAc-P-P-dolichol to GalNAc-P-P-dolichol in eukaryotic cells. Thus, this raises the possibility that in strains where the surface O-antigen containing GalNAc at the reducing termini are involved in a pathological process, O-antigen synthesis could potentially be blocked by inhibiting the bacterial epimerases.
An embodiment of the present invention involves an epimerase that converts GlcNAc-P-P-Und (N-acetylglucosaminylpyrophosphorylundecaprenol) to GalNAc-P-P-Und (N-acetylgalactosaminylpyrophosphorylundecaprenol) in E. coli O157. A still further exemplary aspect of the invention involves the initiation of synthesis of lipid-bound repeating tetrasaccharide having GalNAc at the reducing terminus.
The basis of another aspect of the invention includes the discovery that Campylobacter jejuni contains a general N-linked protein glycosylation system. Various proteins of C. jejuni have been shown to be modified by a heptasaccharide. This heptasaccharide is assembled on undecaprenyl pyrophosphate, the carrier lipid, at the cytoplasmic side of the inner membrane by the stepwise addition of nucleotide activated monosaccharides catalyzed by specific glycosyltransferases. The lipid-linked oligosaccharide then flip-flops (diffuses transversely) into the periplasmic space by a flippase, e.g., PglK. In the final step of N-linked protein glycosylation, the oligosaccharyltransferase (e.g., PglB) catalyzes the transfer of the oligosaccharide from the carrier lipid to asparagine (Asn) residues within the consensus sequence D/E-X-N-Z-S/T, where the X and Z can be any amino acid except Pro. The glycosylation cluster for the heptasaccharide had been successfully transferred into E. coli and N-linked glycoproteins of Campylobacter had been produced.
It had been demonstrated that PglB does not have a strict specificity for the lipid-linked sugar substrate. The antigenic polysaccharides assembled on undecaprenyl pyrophosphate are captured by PglB in the periplasm and transferred to a protein carrier (Feldman, 2005; Wacker, M., et al., Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems. Proc Natl. Acad Sci USA. 2006. 103(18): p. 7088-93.) The enzyme will also transfer a diverse array of undecaprenyl pyrophosphate (UPP) linked oligosaccharides if they contain an N-acetylated hexosamine at the reducing terminus. The nucleotide sequence for pglB and the amino acid sequence for pglB are published at WO2009104074.
Accordingly, one embodiment of the invention involves a recombinant N-glycosylated protein comprising: one or more of an introduced consensus sequence. D/E-X-N-Z-S/T, wherein X and Z can be any natural amino acid except proline; and an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus and N-linked to each of said one or more introduced consensus sequences by an N-glycosidic linkage.
In a further embodiment, the present invention is directed to a recombinant prokaryotic biosynthetic system for producing all or a portion of a polysaccharide comprising an epimerase that synthesizes N-acetylgalactosamine (“GalNAc”) on undecaprenyl pyrophosphate. In a further embodiment, all or a portion of the polysaccharide is antigenic.
In another embodiment, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising: an epimerase that synthesizes GalNAc on undecaprenyl pyrophosphate; and glycosyltransferases that synthesize a polysaccharide having GalNAc at the reducing terminus.
An embodiment of the invention further comprises a recombinant prokaryotic biosynthetic system comprising an epimerase that synthesizes GalNAc on undecaprenyl pyrophosphate and glycosyltransferases that synthesize a polysaccharide, wherein said polysaccharide has the following structure: α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc; and wherein GalNAc is at the reducing terminus of said polysaccharide.
The recombinant prokaryotic biosynthetic system can produce mono-, oligo- or polysaccharides of various origins. Embodiments of the invention are directed to oligo- and polysaccharides of various origins. Such oligo- and polysaccharides can be of prokaryotic or eukaryotic origin. Oligo- or polysaccharides of prokaryotic origin may be from gram-negative or gram-positive bacteria. In one embodiment of the invention, the oligo- or polysaccharide is from E. coli. In a further aspect of the invention, said oligo- or polysaccharide is from E. coli O157. In another embodiment, said oligo- or polysaccharide comprises the following structure: α-D-PerNAc-α-L-Fuc-P-D-Glc-α-D-GalNAc. In a further embodiment of the invention, the oligo- or polysaccharide is from Shigella flexneri. In a still further embodiment, the oligo- or polysaccharide is from Shigella flexneri 6. In a still further aspect, said oligo- or polysaccharide comprises the following structure:
Embodiments of the invention further include proteins of various origins. Such proteins include proteins native to prokaryotic and eukaryotic organisms. The protein carrier can be, for example, AcrA or a protein carrier that has been modified to contain the consensus sequence for protein glycosylation, i.e., D/E-X-N-Z-S/T, wherein X and Z can be any amino acid except proline (e.g., a modified Exotoxin Pseudomonas aeruginosa (“EPA”)). In one embodiment of the invention, the protein is Pseudomonas aeruginosa EPA.
A further aspect of the invention involves novel bioconjugate vaccines having GalNAc at the reducing terminus of the N-glycan. An additional embodiment of the invention involves a novel approach for producing such bioconjugate vaccines that uses recombinant bacterial cells that contain an epimerase which produces GalNAc on undecaprenyl pyrophosphate. In one embodiment, bioconjugate vaccines can be used to treat or prevent bacterial diseases. In further embodiments, bioconjugate vaccines may have therapeutic and/or prophylactic potential for cancer or other diseases.
A typical vaccination dosage for humans is about 1 to 25 μg, preferably about 1 μg to about 10 μg, most preferably about 10 μg. Optionally, a vaccine, such as a bioconjugate vaccine of the present invention, includes an adjuvant.
In an additional embodiment, the present invention is directed to an expression system for producing a bioconjugate vaccine against at least one bacterium comprising: a nucleotide sequence encoding an oligosaccharyl transferase; a nucleotide sequence encoding a protein carrier; at least one polysaccharide gene cluster from the at least one bacterium, wherein the polysaccharide contains GalNAc at the reducing terminus; and a nucleic acid sequence encoding an epimerase. In a further embodiment, the polysaccharide gene cluster encodes an antigenic polysaccharide.
In still a further embodiment, the present invention is directed to an expression system for producing a bioconjugate vaccine against at least one bacterium comprising: a nucleotide sequence encoding an oligosaccharyl transferase; a nucleotide sequence encoding a protein carrier comprising at least one inserted consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; at least one polysaccharide gene cluster from the at least one bacterium, wherein the polysaccharide contains GalNAc at the reducing terminus; and the Z3206 gene. In a further embodiment, the polysaccharide gene cluster encodes an antigenic polysaccharide.
In yet another embodiment, the present invention is directed to a bioconjugate vaccine comprising: a protein carrier; at least one immunogenic polysaccharide chain linked to the protein carrier, wherein said polysaccharide has GalNAc at the reducing terminus, and further wherein said GalNAc is directly linked to the protein carrier; and an adjuvant.
In yet an additional embodiment, the present invention is directed to a bioconjugate vaccine comprising: a protein carrier comprising at least one inserted consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; least one immunogenic polysaccharide from at least one bacterium, linked to the protein carrier, wherein the at least one immunogenic polysaccharide contains GalNAc at the reducing terminus directly linked to the protein carrier; and, optionally, an adjuvant.
Another embodiment of the invention is directed to a method of producing a bioconjugate vaccine, said method comprising: assembling a polysaccharide having GalNAc at the reducing terminus in a recombinant organism through the use of glycosyltransferases; linking said GalNAc to an asparagine residue of one or more target proteins in said recombinant organism, wherein said one or more target proteins contain one or more T-cell epitopes.
In a further embodiment, the present invention is directed to a method of producing a bioconjugate vaccine, said method comprising: introducing genetic information encoding for a metabolic apparatus that carries out N-glycosylation of a target protein into a prokaryotic organism to produce a modified prokaryotic organism; wherein the genetic information required for the expression of one or more recombinant target proteins is introduced into said prokaryotic organism; wherein the genetic information required for the expression of E. coli strain O157 epimerase is introduced into said prokaryotic organism; and wherein the metabolic apparatus comprises glycosyltransferases of a type that assembles a polysaccharide having GalNAc at the reducing terminus on a lipid carrier, and an oligosaccharyltransferase, the oligosaccharyltransferase covalently linking GalNAc of the polysaccharide to an asparagine residue of the target protein, and the target protein containing at least one T-cell epitope; producing a culture of the modified prokaryotic organism; and obtaining glycosylated proteins from the culture medium.
A further aspect of the present invention relates to a pharmaceutical composition. An additional aspect of the invention involves a pharmaceutical composition comprising at least one N-glycosylated protein according to the invention. In light of the disclosure herein, the preparation of medicaments comprising proteins would be well known in the art. A still further aspect of the invention relates to a pharmaceutical composition comprising an antibiotic that inhibits an epimerase that converts GlcNAc-P-P-Und to GalNAc-P-P-Und. In a preferred embodiment, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable excipient, diluent and/or adjuvant.
Suitable excipients, diluents and/or adjuvants are well-known in the art. An excipient or diluent may be a solid, semi-solid or liquid material which may serve as a vehicle or medium for the active ingredient. One of ordinary skill in the art in the field of preparing compositions can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition, and other relevant circumstances (Remington's Pharmaceutical Sciences, Mack Publishing Co. (1990)). The proportion and nature of the pharmaceutically acceptable diluent or excipient are determined by the solubility and chemical properties of the pharmaceutically active compound selected, the chosen route of administration, and standard pharmaceutical practice. The pharmaceutical preparation may be adapted for oral, parenteral or topical use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like. The pharmaceutically active compounds of the present invention, while effective themselves, can be formulated and administered in the form of their pharmaceutically acceptable salts, such as acid addition salts or base addition salts, for purposes of stability, convenience of crystallization, increased solubility, and the like.
In instances where specific nucleotide or amino acid sequences are noted, it will be understood that the present invention encompasses homologous sequences that still embody the same functionality as the noted sequences. In an embodiment of the invention, such sequences are at least 85% homologous. In another embodiment, such sequences are at least 90% homologous. In still further embodiments, such sequences are at least 95% homologous.
The determination of percent identity between two nucleotide or amino acid sequences is known to one of skill in the art.
Nucleic acid sequences described herein, such as those described in the sequence listing below, are examples only, and it will be apparent to one of skill in the art that the sequences can be combined in different ways. Additional embodiments of the invention include variants of nucleic acids. A variant of a nucleic acid (e.g., a codon-optimized nucleic acid) can be substantially identical, that is, at least 80% identical, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical, to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. Nucleic acid variants of a sequence that contains SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29 include nucleic acids with a substitution, variation, modification, replacement, deletion, and/or addition of one or more nucleotides (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 nucleotides) from a sequence that contains SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or parts thereof.
For example, in an embodiment of the instant invention, such variants include nucleic acids that encode an epimerase which converts GlcNAc-P-P-Und to GalNAc-P-P-Und and that i) are expressed in a host cell, such as, for example, E. coli and ii) are substantially identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, or parts thereof.
Nucleic acids described herein include recombinant DNA and synthetic (e.g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. In the case of single-stranded nucleic acids, the nucleic acid can be a sense strand or antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives.
Plasmids that include a nucleic acid described herein can be transfected or transformed into host cells for expression. Techniques for transfection and transformation are known to those of skill in the art.
All publications mentioned herein are incorporated by reference in their entirety. It is to be understood that the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination. As used herein, unless the context clearly dictates otherwise, references to the singular, such as the singular forms “a,” an,” and “the,” include the plural, and references to the plural include the singular.
The invention is further defined by reference to the following examples that further describe the compositions and methods of the present invention, as well as its utility. It will be apparent to those skilled in the art that modifications, both to compositions and methods, may be practiced which are within the scope of the invention.
EXAMPLES Bacterial Strains and Plasmids E. coli strains PR4019 (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322) and PR21546 (Meier-Dieter, U., Starman, R., Barr, K., Mayer, H. and Rick, P. I). (1990) J. Biol. Chem., 265, 13490-13497) were generous gifts from Dr. Paul Rick, Bethesda, Md., and E. coli O157:H45 (Stephan, R., Borel, N., Zweifel, C., Blanco, M, and Blanco, J. E. (2004) BMC Microbiol 4:10) was a gift from Dr. Claudio Zweifel, Veterinary Institute, University of Zurich, E. coli DH5α (Invitrogen) was used as the host for cloning experiments and for protein glycosylation analysis. Plasmids used are listed in Table 2.
TABLE 2
Plasmids used in Examples
Plasmid Description Ref
pMLBAD Cloning vector, TmpR Lefebre &
Valvano
(2002)
pMLBAD:Z3206 Z3206 in pMLBAD, TmpR, expression Examples
(SEQ ID NO: 23) controlled by arabinose-inducible herein
promoter
pMLBAD:gne gne in pMLBAD, TmpR, expression Examples
(SEQ ID NO: 24) controlled by arabinose-inducible herein
promoter
pACYCpgl C. jejuni pgl cluster CmR Wacker,
et al.
(2002)
pACYCgne::kan C. jejuni pgl cluster containing a kan Linton, et
cassette in gne, CmR, KanR al. (2005)
pWA2 Soluble periplasmic hexa-His-tagged Feldman,
AcrA under control of Tet promoter in et al.
pBR322, AmpR (2005)
Materials—
[1,6-3H]GlcNAc (30 Ci/mmol), UDP-[1-3H]GlcNAc (20 Ci/mmol) and UDP-[6-3H]GalNAc (20 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, Mo.). Quantum 1 silica gel G thin layer plates are a product of Quantum Industries (Fairfield, N.J.), and Baker Si250 Silica Gel G plates are manufactured by Mallinekrodt Chemical Works. Yeast extract and Bacto-peptone were products of BD Biosciences. All other chemicals were obtained from standard commercial sources. Trimethoprim (50 μg/ml), chloramphenicol (20 μg/ml), ampicillin (100 μg/ml), and kanamycin (50 μg/ml) were added to the media as needed.
Construction of Recombinant Plasmids—
E. coli strain DH5α was used for DNA cloning experiments and constructed plasmids were verified by DNA sequencing. The Z3206 gene was amplified from E. coli O157:H45 by PCR with oligonucleotides Z3206-Fw and Z3206-RvHA (AAACCCGGGATGAACGATAACG TTTTGCTC (SEQ ID NO: 17) and AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTACTCAGAAACAA ACGTTATGTC (SEQ ID NO: 18): restriction sites are underlined). The PCR fragment was digested with SmaI and XbaI and ligated into SmaI-XbaI cleaved pMLBAD vector (Lefebre, M. D. and Valvano M. A. (2002) Appl Environ Microbiol 68: 5956-5964). This resulted in plasmid pMLBAD:Z3206 (SEQ ID NO: 23) encoding Z3206 with a C-terminal hemagglutinin tag.
The gne gene was amplified from pACYCpgl (Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wrenn, B. W., Aebi, M. (2002) Science 298, 1790-1793), encoding Campylobacter jejuni pgl cluster, with oligonucleotides gne-Fw and gne-RV (AAACCATGGATGAAAATTCTTATTAGCGG (SEQ ID NO: 19) and AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTAGCACTGTTTTTC CCAATC (SEQ ID NO: 20); restriction sites are underlined). The PCR product was digested with NcoI and XbaI and ligated into the same sites of pMLBAD to generate plasmid pMLBAD:gne (SEQ ID NO: 24) which encodes One with a C-terminal hemagglutinin tag (Table 2).
Growth Conditions, Protein Expression and Immunodetection—
E. coli strains were cultured in Luria-Bertani medium (1% yeast extract, 2% Bacto-peptone, 0.6% NaCl) at 37° C. with vigorous shaking. Arabinose inducible expression was achieved by adding arabinose at a final concentration of 0.02-0.2% (w/v) to E. coli cells grown up to an A600 of 0.05-0.4. The same amount of arabinose was added again 5 h post-induction, and incubation continued for 4-15 h.
Analytical Procedures—
Protein concentrations were determined using the BCA protein assay (Pierce) after precipitation of membrane proteins with deoxycholate and trichloroacetic acid according to the Pierce Biotechnology bulletin “Eliminate Interfering Substances from Samples for BCA Protein Assay.” Samples were analyzed for radioactivity by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer after the addition of 0.5 ml of 1% SDS and 4 ml of Econosafe Economical Biodegradable Counting Mixture (Research Products International, Corp., Mount Prospect, Ill.).
Example 1 Identification of an E. coli O157 Gene Encoding GlcNAc-P-P-Und 4-Epimerase We describe herein the surprising discovery of a new biosynthetic pathway in which GalNAc-P-P-Und is formed by the epimerization of the 4-OH of GlcNAc-P-P-Und catalyzed by the previously unknown action of a 4-epimerase. In this pathway, GlcNAc-P-P-Und is formed by the transfer of GlcNAc-P from UDP-GlcNAc, catalyzed by WecA, and then GlcNAc-P-P-Und is epimerized to GalNAc-P-P-Und by GlcNAc-P-P-Und-4-epimerase, which was a previously unknown pathway (FIG. 2.
The gene encoding a candidate for the GlcNAc-P-P-Und 4-epimerase was identified by DNA homology searches. Homology searches were performed using the U.S. National Library of Medicine databases found at http:blast.ncbi.nlm.nih.govBlast.cgi. Genomic sequences of different bacteria encoding O antigen repeating units having a GalNAc at the reducing terminus were screened. One group with a repeating unit containing a GalNAc at the reducing terminus, and a second group lacking a terminal GalNAc in the repeating unit were compared to identify potential epimerases. Using these criteria, Z3206 was identified as a candidate GlcNAc-P-P-Und 4-epimerase (Table 1).
The GlcNAc 4-epimerase genes present in E. coli strains with O-antigen repeat units containing GalNAc can be separated into two homology groups as shown in Table 1. It was surprisingly discovered that one homology group (containing grid) clearly was correlated with the presence of GalNAc as the initiating sugar on the O-antigen repeat unit. It was further surprisingly discovered that the second group (containing gne2) exhibits a high degree of similarity to the UDP-Glc epimerase, GalE, and is found in E. coli strains that do not initiate O-antigen repeat unit synthesis with GalNAc. Z3206 in E. coli O157, a gene with a high degree of homology to gne1, was identified as a candidate GlcNAc-P-P-Und 4-epimerase. The genomic location of the Z3206 gene is consistent with a role in this pathway, as it resides between galF of the O-antigen cluster and wcaM which belongs to the colanic acid cluster.
The research described in Examples 2-11 further confirms the above discoveries, including identifying the GlcNAc 4-epimerase (E. coli O157 Z3206) as catalyzing the formation of GalNAc-P-P-Und.
Example 2 UDP-GalNAc is not a Substrate for E. coli WecA (GlcNAc-phosphotransferase) To determine if E. coli WecA will utilize UDP-GalNAc as a GalNAc-P donor to form GalNAc-P-P-Und, membrane fractions from E. coli strains K12, PR4019, a WecA-overexpressing strain, and O157, which synthesize a tetrasaccharide O-antigen repeat unit with GalNAc at the reducing terminus presumably initiated by the synthesis of GalNAc-P-P-Und, were incubated with UDP-[3H]GalNAc.
Preparation of E. coli Membranes—
Bacterial cells were collected by centrifugation at 1,000×g for 10 min, washed once in ice-cold phosphate-buffered saline, once with cold water, and once with 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose. The cells were resuspended to a density of ˜200 A600 units/ml in 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 10 mM EDTA containing 0.2 mg/ml lysozyme, and incubated at 30° C. for 30 min. Bacterial cells were recovered by centrifugation at 1,000×g for 10 min, quickly resuspended in 40 volumes of ice-cold 10 mM Tris-HCl, pH 7.4, and placed on ice. After 10 min the cells were homogenized with 15 strokes with a tight-fitting Dounce homogenizer and supplemented with 0.1 mM phenylmethylsulfonyl fluoride and sucrose to a final concentration of 0.25 M. Unbroken cells were removed by centrifugation at 1,000×g for 10 min, and cell envelopes were recovered by centrifugation at 40,000×g for 20 min. The membrane fraction was resuspended in 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA and again sedimented at 40,000×g and resuspended in the same buffer to a protein concentration of ˜20 mg/ml. Membrane fractions were stored at −20° C. until needed.
Assay for the Biosynthesis of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und in E. coli Membranes In Vitro—
Reaction mixtures for the synthesis of GlcNAc-P-P-Und and GalNAc-P-P-Und contained 50 mM Tris-HCl, pH 8, 40 mM MgCl2, 5 mM dithiothreitol, 5 mM 5′ AMP. E. coli membrane fraction (50-200 μg membrane protein, and either 5 μm UDP-[3H]GlcNAc/GalNAc (500-2500 dpm/pmol) in a total volume of 0.05 ml, After incubation at 37° C., reactions were terminated by the addition of 40 volumes of CHCl3/CH3OH (2:1), and the total lipid extract containing [3H]HexNAc-P-P-undecaprcnols was prepared as described previously (Waechter. C. J., Kennedy, J. L. and Harford, J. B. (1976) Arch. Biochem, Biophys. 174, 726-737). After partitioning, the organic phase was dried under a stream of nitrogen and redissolved in 1 ml CHCl3/CH3OH (2:1), and an aliquot (0.2 ml) was removed, dried in a scintillation vial, and analyzed for radioactivity by liquid scintillation spectrometry in a Packard Tri-Carb 2100 TR liquid scintillation specometer. To determine the rate of synthesis of [3H]GlcNAc-P-P-Und or [3H]GalNAc-P-P-Und, the lipid extract was dried under a stream of nitrogen, redissolved in a small volume of CHCl3/CH3OH (2:1), and spotted on a 10×20-cm borate-impregnated Baker Si250 silica gel plate, and the plate was developed with CHCl3, CH3OH, H2O, 0.2 M sodium borate (65:25:2:2). Individual glycolipids were detected with a Bioscan AR2000 Imaging Scanner (Bioscan, Washington, D.C.). The biosynthetic rates for each glycolipid were calculated by multiplying the total amount of radioactivity in [3H]GlcNAc/GalNAc-P-P-Und by the percentage of the individual [3H] glycolipids.
Membrane fractions from different E. coli strains (K12, PR4019 and O157) were incubated with either UDP-[3H]GlcNAc or UDP-[3H]GalNAc and the incorporation into [3H]GlcNAc/GalNAc-P-P-Und was determined as described above. As seen in Table 3, no labeled glycolipids were detected after the incubation with UDP-[3H]GalNAc, only GlcNAc-P-P-Und was detectable when membrane fractions were incubated with UDP-[3H]GlcNAc
TABLE 3
Synthesis of [3H]GlcNAc/GalNAc-P-P-undecaprenol in E. coli membrane
fractions using either UDP-[3H]GlcNAc or UDP-[3H]GalNAc as substrate
[3H]Glycolipid formed
Source of Sugar nucleotide GlcNAc-P-P-Und GalNAc-P-P-Und
membranes added (pmol/mg) (pmol/mg)
K12 UDP-[3H]GlcNAc 6.4 <0.01
K12 UDP-[3H]GalNAc <0.01 <0.01
PR4019 UDP-[3H]GlcNAc 44 <0.01
PR4019 UDP-[3H]GalNAc <0.01 <0.01
O157 UDP-[3H]GlcNAc 1.5 0.5
O157 UDP-[3H]GalNAc <0.01 <0.01
Moreover, neither the addition of exogenous Und-P to incubations with membranes from PR4019, the WecA-overexpressing strain, or the addition of cytosolic fractions from O157 cells resulted in the formation of GalNAc-P-P-Und from UDP-GalNAc. These results demonstrate that UDP-GalNAc is not a substrate for WecA and suggest that GalNAc-P-P-Und is formed by an alternative mechanism.
When membranes from strain K12 were incubated with UDP-[3H]GlcNAc, [3H]GlcNAc-P-P-Und was synthesized as expected (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322). However, when membranes from strain O157 were incubated with UDP-[3H]GlcNAc, in addition to [3H]GlcNAc-P-P-Und, a second labeled lipid shown to be [3H]GalNAc-P-P-Und (see below) was observed. When the time course for the formation of the two glycolipids was examined, the incorporation of radioactivity into [3H]GlcNAc-P-P-Und (FIG. 1, O) occurred more quickly and to a higher extent than into [3H]GalNAc-P-P-Und (FIG. 1, ), compatible with a precursor-product relationship (FIG. 2).
The observation that E. coli O157 membranes do not utilize UDP-GalNAc as a GalNAc-P donor for the synthesis of GalNAc-P-P-Und is one example which confirms the biosynthetic pathway for the formation of GalNAc-P-P-Und illustrated in FIG. 2. In this scheme, GlcNAc-P-P-Und is formed by the transfer of GlcNAc-P from UDP-GlcNAc, catalyzed by WecA, and then GlcNAc-P-P-Und is epimerized by the action of a previously unknown 4-epimerase to produce GalNAc-P-P-Und.
Example 3 Characterization of [3H]GalNAc-P-P-Und Formed In Vitro with Membrane Fractions from E. coli Strain O157 Consistent with the additional O157-specific glycolipid product detected in FIG. 1, as GalNAc-P-P-Und, it was stable to mild alkaline methanolysis (toluene/methanol 1:3, containing 0.1 N KOH, 0° C., 60 min), retained by DEAE-cellulose equilibrated in CHCl3/CH3OH/H2O (10:10:3), and eluted with CHCl3/CH3OH/H2O (10:10:3) containing 20 mM ammonium acetate as reported previously for [3H]GlcNAc1-2-P-P-Dol (Waechter, J. and Harford, B. (1977) Arch. Biochem. Biophys. 181, 185-198).
[3H]GalNAc-P-P-Und was clearly resolved from [3H]GalNAc-P-P-Und by thin layer chromatography on borate-impregnated silica gel G (Kean, E. L. (1966) J. Lipid Res. 7,149-452) and purified by preparative TLC as shown in FIG. 3A and FIG. 3B.
Preparation of Borate-Impregnated Thin Layer Plates and Whatman No. 1 Paper—
Silica gel thin layer plates were impregnated with sodium borate by briefly immersing the plates in 2.5% Na2B4O7.10 H2O in 95% methanol as described by Kean (Kean, E. L. (1966) J. Lipid Res. 7.449-452). The borate-impregnated TLC plates were dried overnight at room temperature and stored in a vacuum dessicator over Drierite until use. Immediately before chromatography, the plates were activated by heating briefly (˜10-15 min) to 100° C. Whatman No. 1 paper was impregnated with sodium borate by dipping 20×30-cm sheets of Whatman 1 paper in 0.2 M Na2B4O7.10H2O. The Whatman No. 1 paper sheets were pressed firmly between two sheets of Whatman No. 3MM paper and allowed to dry at room temperature for several days, as described by Cardini and Leloir (Cardini, C. E. and Leloir, L. F. (1957) J. Biol. Chem. 225, 317-324).
Characterization of Glycan Products Formed in In Vitro Reactions—
The glycans of the individual glycolipids ([3H]GalNAc-P-P-Und and [3H]GlcNAc-P-P-Und) were characterized by descending paper chromatography after release by mild acid hydrolysis. The GlcNAc/GalNAc lipids were dried under a stream of nitrogen in a conical screw-cap tube and heated to 100° C., 15 min in 0.2 ml 0.01 M HCl. After hydrolysis the samples were applied to a 0.8-ml mixed-bed ion-exchange column containing 0.4 ml of AG50WX8 (H+) and 0.4 ml AG1X8 (acetate form) and eluted with 1.5 ml water. The eluate was dried under a stream of nitrogen, redissolved in a small volume of H2O (0.02 ml), spotted on a 30-cm strip of borate-impregnated Whatman No. 1 paper, and developed in descending mode with butanol/pyridine/water (6:4:3) for 40-50 h. After drying, the paper strips were cut into 1-cm zones and analyzed for radioactivity by scintillation spectrometry. GlcNAc and GalNAc standards were detected using an aniline-diphenylamine dip reagent (Schwimmer, S. and Benvenue, A. (1956) Science 123, 543-544).
Glycan products were converted to their corresponding alditols by reduction with 0.1 M NaBH4 in 0.1 M NaOH (final volume ml) following mild acid hydrolysis as described above. After incubation at room temperature overnight, the reactions were quenched with several drops of glacial acetic acid and dried under a stream of nitrogen out of methanol containing 1 drop of acetic acid, several times. The alditols were dissolved in water, desalted by passage over 0.5 ml columns of AG50WX8 (H+) and AG1X8 (acetate), dried under nitrogen, and spotted on 30-cm strips of Whatman No. 3MM paper. The Whatman No. 3 MM strips were developed overnight in descending mode with ethyl acetate, pyridine, 0.1 M boric acid (65:25:20), dried, cut into 1-cm zones, and analyzed for radioactivity by scintillation spectrometry. GlcNAcitol and GalNAcitol standards were visualized using a modification of the periodate-benzidine dip procedure (Gordon, H. T., Thornburg, W. and Werum, L. N. (1956) Anal. Chem. 28, 849-855). The paper strips were dipped in acetone, 0.1 M NaIO4 (95:5), allowed to air dry for 3 min, and then dipped in acetone/acetic acid/H2O/o-tolidine (96:0.6:4.4:0.2 gm), Alditols containing cis-diols stain as yellow spots on a blue background.
Mass Spectrometry (“MS”) of Glycolipids—
Purified glycolipids were analyzed using an ABI/MDS Sciex 4000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometer with an ABI Turbo V electrospray ionsource (ABIMDS-Sciex, Toronto, Canada). In brief, samples were infused at 10 μl/min with ion source settings determined empirically, and MS/MS (mass spectroscopy in a second dimension) information was obtained by fragmentation of the molecular ion in linear ion trap mode.
When the glycolipid was treated with mild acid (0.01 N HCl, 100° C., 15 min), the water-soluble product co-chromatographed with [3H]GalNAc on descending paper chromatography with borate-impregnated Whatman No. 1 paper (FIG. 3C). In addition, when the labeled sugar was reduced, it was converted to [3H]alditol, GalNAc-OH (FIG. 3D). Moreover, negative-ion MS analysis yielded the [M-H]-ion of m/z=1128, expected for GalNAc-P-P-Und, and the MS/MS daughter ion spectrum showed a prominent ion at m/z=907, expected for a glycolipid containing P-P-Und (Guan, Z., Breazeale, S. D. and Raetz, C. R. (2005) Anal. Biochem. 345, 336-339). The identification of the glycolipid product formed by strain O157 as GalNAc-P-P-Und is also supported by its formation from exogenous GlcNAc-P-P-Und (see Example 7).
Example 4 Metabolic Labeling of [3H]GalNAc-P-P-Und (In Vivo) with [3H]GlcNAc in E. coli Cells Expressing the Z3206 Gene To investigate whether expression of the E. coli O157 Z3206 gene enabled cells to synthesize GalNAc-P-P-Und, E. coli strain 21546 (Meier-Dieter, U., Starman, R., Barr, K., Mayer, H. and Rick, P. D. (1990) J. Biol. Chem., 265, 13490-13497) expressing the Z3206 gene was labeled metabolically with [3H]GlcNAc and analyzed for [3H]GlcNAc/GalNAc-P-P-Und formation.
Metabolic Labeling of Bacterial Cells—
E. coli cells were cultured with vigorous shaking in Luria-Bertani medium at 37° C. to an A600 of 0.5-1. [3H]GlcNAc was added to a final concentration of 1 μCi/ml and the incubation was continued for 5 min at 37° C. The incorporation of radiolabel into glycolipids was terminated by the addition of 0.5 gm/ml crushed ice, and the cultures were thoroughly mixed. The bacterial cells were recovered by centrifugation at 4000×g for 10 min, and the supernatant was discarded. The cells were washed with ice-cold phosphate-buffered saline two times, resuspended by vigorous vortex mixing in 10 volumes (cell pellet) of methanol, and sonicated briefly with a probe sonicator at 40% full power. After sonication, 20 volumes of chloroform were added, and the extracts were mixed vigorously and allowed to stand at room temperature for 15 min. The insoluble material was sedimented by centrifugation, and the pellet was re-extracted with a small volume of CHCl3/CH3OH (2:1) twice. The combined organic extracts were then processed as described below.
Purification of GlcNAc-P-P-Und and GalNAc-P-P-Und—
GlcNAc/GalNAc-P-P-Und was extracted with CHCl3/CH3OH (2:1) and freed of water-soluble material by partitioning as described elsewhere (Waechter, C. J., Kennedy, J. L. and Harford, J. B. (1976) Arch. Biochem. Biophys. 174, 726-737). The organic extract was then dried under a stream of nitrogen, and the bulk glycerophospholipids were destroyed by deacylation in toluene/methanol (1:3) containing 0.1 N KOH at 0° C. for 60 min. The deacylation reaction was neutralized with acetic acid, diluted with 4 volumes of CHCl3/CH3OH (2:1), and washed with 15 volume of 0.9% NaCl. The organic (lower) phase was washed with 13 volume of CHCl3, CH3OH, 0.9% NaCl (3:48:47), and the aqueous phase was discarded. The organic phase was diluted with sufficient methanol to accommodate the residual aqueous phase in the organic phase and applied to a DEAE-cellulose column (5 ml) equilibrated with CHCl3/CH3OH (2:1). The column was washed with 20 column volumes of CHCl3/CH3OH/H2O (10:10:3) and then eluted with CHCl3/CH3OH/H2O (10:10:3) containing 20 mM ammonium acetate. Fractions (2 ml) were collected and monitored for either radioactivity, or GlcNAc/GalNAc-P-P-Und using an anisaldehyde spray reagent (Dunphy, P. J., Kerr, J. D., Pennock, J. F., Whittle, K. J., and Feeney, J. (1967) Biochim. Biophys. Acta 136, 136-147) after resolution by thin layer chromatography on borate-impregnated silica plates (as described earlier).
E. coli strain 21546 was selected as the host for the Z3206 expression studies because a mutation in UDP-ManNAcA synthesis results in a block in the utilization of GlcNAc-P-P-Und for the synthesis of the enterobacterial common antigen. Because E. coli 21546 is derived from E. coli K12 it does not synthesize an O-antigen repeat as well (Stevenson, G., Neal, B., Liu, D., Hobbs, M., Packer, N. H., Batley, M., Redmond, J. W., Lindquist, L. and Reeves, P. (1994) J. Bacterial., 176, 4144-4156), and thus, larger amounts of GlcNAc-P-P-Und accumulate for the conversion to GalNAc-P-P-Und. When strain 21546 and the transformant expressing the Z3206 gene were labeled with [3H]GlcNAc and the radiolabeled lipids were analyzed by thin layer chromatography on borate-impregnated silica gel plates, the parental strain (FIG. 4A) synthesized only one labeled lipid, GlcNAc-P-P-Und. However, 21546 cells expressing the Z3206 gene (FIG. 4B) also synthesized an additional labeled lipid shown to be GalNAc-P-P-Und.
Example 5 Membrane Fractions from E. coli Cells Expressing the Z3206 Gene Synthesize GalNAc-P-P-Und In Vitro To corroborate that the protein encoded by the E. coli O157 Z3206 gene catalyzed the synthesis of GalNAc-P-P-Und, membrane fractions from E. coli cells expressing the Z3206 gene were incubated with [3H]UDP-GlcNAc and the [3H]glycolipid products were analyzed by thin layer chromatography (chromatographic preparation and characterization methods are described in Example 3) on borate-impregnated silica gel plates as shown in FIG. 5. When membrane fractions from E. coli K12 or the host strain E. coli 21546 cells were incubated with UDP-[3H]GlcNAc, only [3H]GlcNAc-P-P-Und was observed (FIG. 5A and FIG. 5C). However, membrane fractions from E. Coli O157 and E. coli 21546 expressing Z3206 formed GalNAc-P-P-Und as well (FIG. 5B and FIG. 5D).
Example 6 Formation of GlcNAc-P-P-Und, but not GalNAc-P-P-Und, is Reversed in the Presence of UMP To provide additional evidence that GalNAc-P-P-Und is synthesized from GlcNAc-P-P-Und, and not by the action of WecA using UDP-GalNAc as a glycosyl donor, the effect of discharging endogenous, pre-labeled [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und with UMP was examined. The GlcNAc-phosphotransferase reaction catalyzed by WecA is freely reversible by the addition of excess UMP re-synthesizing UDP-GlcNAc and releasing Und-P.
In this experiment membrane fractions from E. coli strain 21546 expressing Z3206 were pre-labeled for 10 min with UDP-[3H]GlcNAc followed by the addition of 1 mM UMP, and the amount of each labeled glycolipid remaining was determined. The results illustrated in FIG. 6A show the relative amounts of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und at the end of the 10 min labeling period. After incubation with 1 mM UMP for 1 min it can be seen that there is a substantial loss of [3H]GalNAc-P-P-Und, whereas the [3H]GalNAc-P-P-Und peak is relatively unchanged (FIG. 6B) (chromatographic preparation and characterization methods are described in Example 5), This observation is consistent with the results in Table 3 indicating that WecA does not catalyze the transfer of GalNAc-P into GalNAc-P-P-Und from UDP-GalNAc. It is noteworthy that during the second minute of incubation with UMP (FIG. 6C), the loss of GlcNAc-P-P-Und slows, and there is a slight reduction in the peak of [3H]GalNAc-P-P-Und, suggesting that [3H]GalNAc-P-P-Und is re-equilibrating with the [3H]GlcNAc-P-P-Und pool by reversal of the epimerase reaction (see Example 7).
Example 7 Interconversion of Exogenous, Purified [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und Catalyzed by Membranes from E. Coli Cells Expressing Z3206 To provide direct evidence that GlcNAc-P-P-Und and GalNAc-P-P-Und can be directly interconverted by membrane fractions from E. coli cells expressing Z3260, purified [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were tested as exogenous substrates.
Purified [3H]GlcNAc-P-P-Und/[3H]GalNAc-P-P-Und were prepared as in Example 4 (Metabolic Labeling of Bacterial Cells and Purification of GlcNAc-P-P-Und and GalNAc-P-P-Und). [3H]HexNAc-P-P-undecaprenols (2000 dpm/pmol, dispersed in 1% Triton X-100, final concentration 0.1%) were incubated with E. coli membranes as in Example 2 in Assay For the Biosynthesis of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und in E. coli Membranes In Vitro.
Preliminary experiments showed that the epimerase was active when exogenous [3H]GalNAc-P-P-Und was added to the reaction mixtures dispersed in Triton X-100, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid), Nonidet P-40, or octylglucoside and exhibited a pH optimum in the range 7-8.5. The chromatographic mobility of the purified [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und before incubation with membrane fractions is shown in FIG. 7A and FIG. 7D. As seen in FIG. 7B and FIG. 7E, the glycolipids are unaffected by incubation with membrane fractions from E. coli 21546. However, incubation of the purified glycolipids with membrane fractions from E. coli 21546 expressing Z3206 catalyzes the conversion of exogenous [3H]GlcNAc-P-P-Und to [3H]GalNAc-P-P-Und (FIG. 7C) and the conversion of [3H]GalNAc-P-P-Und to [3H]GlcNAc-P-P-Und (FIG. 7F). These results demonstrate directly that GlcNAc-P-P-Und and GalNAc-P-P-Und can be enzymatically interconverted in E. coli strains expressing the Z3206.
Example 8 E. coli Z3206 is not a UDP-GlcNAc 4-Epimerase To determine if Z3206 can catalyze the formation of UDP-GalNAc, the N-glycosylation apparatus from C. jejuni was expressed in E. coli. In this reporter system, glycosylation of the target protein AcrA is dependent on the presence of the pgl locus (Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wrenn, B. W., Aebi, M. (2002) Science 298, 1790-1793), including a functional Gne UDP-Glc/UDP-GlcNAc epimerase (Bernatchez, S., Szymanski, C. M., Ishiyama, N., Li, J., Jarrell, H. C., Lau, P. C., Berghuis, A. M., Young, N. M., Wakarchuk, W. W. (2005) J. Biol. Chem. 280, 4792-4802). Glycosylation of AcrA is lost if the pgl cluster contains a deletion of gne (Linton, D., Dorrell, N., Hitchen, P. G., Amber, S., Karlyshev, A. V., Morris, H. R., Dell, A., Valvano, M. A., Aebi, M. and Wren, B. W. (2005) Mol Microbiol. 55, 1695-1703). The ability of Z3206 to restore AcrA-glycosylation in the presence of the pgl operon Δgne was investigated in vivo by expressing AcrA (pWA2) together with the pgl locus Δgne complemented by either Gne (pMLBAD:gne) or Z3206 (pMLBAD:Z3206).
Total E. coli cell extracts were prepared for immunodetection analysis using cells at a concentration equivalent to 1 A600 unit that were resuspended in 100 μl of SDS loading buffer (Laemmli, U. (1970) Nature 227, 680-685). Aliquots of 10 μl were loaded on 10% SDS-PAGE. Periplasmic extracts of E. coli cells were prepared by lysozyme treatment (Feldman, M. F., Wacker, M., Hernandez, M., Hitchen, P. G., Marolda, C. L., Kowarik, M., Morris, H. R., Dell, A., Valvano, M. A., Aebi, M. (2005) Proc Natl Acad Sci USA 102, 3016-3021), and 10 μl of the final sample (corresponding to 0.2 A600 units of cells) was analyzed by SDS-PAGE. After being blotted on nitrocellulose membrane, sample was immunostained with the specific antiserum (Aebi, M., Gasscnhuber, J., Domdey, H., and te Heesen, S. (1996) Glycobiology 6, 439-444). Anti-AcrA (Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wrenn, B. W., Aebi, M. (2002) Science 298, 1790-1793) antibodies were used. Anti-rabbit IgG-HRP (Bio-Rad) was used as secondary antibody. Detection was carried out with ECL™ Western blotting detection reagents (Amersham Biosciences).
As shown in FIG. 8, the glycosylated protein, which migrates slower than the unglycosylated form, was formed only when cells expressing pgl locus Δgne were complemented by One (lane 2). Z3206 was unable to restore glycosylation of the reporter glycoprotein (FIG. 8, lane 1). Accordingly, Z3206 does not complement glycosylation of AcrA in a Gne dependent glycosylation system. Expression of Gne and membrane-associated Z3206 were confirmed by immunodctection.
Example 9 Analysis of S. flexneri 6+/− Z3206 LPS In FIG. 9 are depicted some of the genes required for the biosynthesis of the Shigella flexneri 6 O-antigen: genes encoding enzymes for biosynthesis of nucleotide sugar precursors; genes encoding glycosyltransferases; genes encoding O antigen processing proteins; and genes encoding proteins responsible for the O-acetylation. The structure of the O antigen has been elucidated by Dmitriev, B. A. et al (Dmitriev. B. A., et al Somatic Antigens of Shigella Eur J Biochem, 1979. 98: p. 8; Liu B et al Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27).
To identify all the genes required for the biosynthesis of the Shigella flexneri 6 O-antigen a genomic library was constructed.
Cloning of S. flexneri 6 genomic DNA—
S. flexneri 6 genomic DNA was isolated using a Macherey-Nagel NucleoSpin® Tissue Kit following the protocol for DNA isolation from bacteria. DNA was isolated from five S. flexneri 6 overnight cultures at 2 ml each and final elution was done with 100 μl elution buffer (5 mM Tris/HCl, pH 8.5). The eluted fractions were pooled, precipitated by isopropanol and the final pellet was resuspended in 52 μl TE buffer of which the total volume was subjected to end-repair according to the protocol given by CopyControl™ Fosmid Library Production Kit (EPICENTRE). End-repaired DNA was purified on a 1% low melting point agarose gel run with 1×TAE buffer, recovered and precipitated by ethanol as described in the kit protocol. Resuspension of the precipitated DNA was done in 7 μl TE buffer of which 0.15 μl DNA was ligated into pCC1FOS (SEQ ID NO: 27) according to the EPICENTRE protocol. Packaging of the ligation product into phage was performed according to protocol and the packaged phage was diluted 1:1 in phage dilution buffer of which 10 μl were used to infect 100 μl EPI300-T1 cells that were previous grown as described by EPICENTRE. Cells (110 μl) were plated six times with approximately 100 colonies per plate such that the six plates contain the entire S. flexneri 6 genomic library. Plates were developed by colony blotting and positive/negative colonies were western blotted and silver stained.
Colony Blotting—
For colony blots a nitrocellulose membrane was laid over the solid agar plate, removed, washed three times in 1×PBST and treated in the same manner. The membrane was first blocked in 10% milk for one hour at room temperature after which it was incubated for one hour at room temperature in 2 ml 1% milk (in PBST) with the anti-type VI antiserum (primary antibody). After three washes in PBST at 10 minutes each, the membrane was incubated for another hour at room temperature in the secondary antibody, 1:20000 peroxidase conjugated goat-anti-rabbit IgG (BioRad) in 2 ml 1% milk (in PBST). After a final three washes with PBST (10 minutes each) the membrane was developed in a UVP Chemi Doc Imaging System with a 1:1 mix of luminol and peroxide buffer provided by the SuperSignal® West Dura Extended Duration Substrate Kit (Thermo Scientific).
The clone reacting with S. flexneri 6 antiserum following production of a S. flexneri 6 genomic library was sequenced by primer walking out of the region previously sequenced by Liu et al. (Liu et al., 2008) reaching from rmlB to wtbZ (FIG. 9). Primers rmlB_rev and wfbZ_fwd (S. flexneri—Z3206) annealed in rmlB and wfbZ and were used to sequence the insert of the clone until wcaM and hisI/F were reached (S. flexneri+Z3206), respectively (FIG. 10).
In order to establish whether O antigen synthesis is maintained in clones lacking Z3206 (thus hindering epimerization of und-GlcNAc to und-GalNAc), two plasmids were constructed (SEQ ID NO. 28 and SEQ ID NO. 29) (FIG. 10), transformed into E. coli cells and analyzed by silver staining and western blot.
As shown in FIG. 11, LPS is produced in E. coli cells + or − Z3206, The O antigen can be produced without Z3206 however with lower production yield, which indicates that the efficiency of polysaccharide production without the epimerase (Z3206) is lower.
Example 10 Analysis of S. flexneri 6+/− Z3206 LLO Purification of undecaprenol-PP-O antigen by C18 column chromatography—
E. coli cells expressing S. flexeneri antigen+/− Z3206 were pelleted, washed once in 50 ml 0.9% NaCl and the final pellets were lyophilized overnight. The pellets were washed once in 30 ml 85-95% methanol, reextracted with 10:10:3 chloroform-methanol-water (v/v/v) and the extracts were converted to a two-phase Bligh/Dyer system by addition of water, resulting in a final ratio of 10:10:9 (C:M:W). Phases were separated by centrifugation and the upper aqueous phases were loaded each on a C18 Sep-Pak cartridge conditioned with 10 ml methanol and equilibrated with 10 ml 3:48:47 (C:M:W). Following loading, the cartridges were washed with 10 ml 3:48:47 (C:M:W) and eluted with 5 ml 10:10:3 (C:M:W). 20 OD samples of the loads, flow-throughs, washes and elutions of the C18 column were dried in an Eppendorf Concentrator Plus, washed with 250 μl methanol, reevaporated and washed a further three times with 30 μl ddH2O.
Glycolipid Hydrolysis
The glycolipid samples from the wash of the C18 column were hydrolysed by dissolving the dried samples in 2 ml n-propanol:2 M trifluoroacetic acid (1:1), heating to 50° C. for 15 minutes and evaporating to dryness under N2.
Oligosaccharide labeling with 2-aminobenzoate and HPLC Labeling was done according to Bigge et al. (Bigge, 1995) and glycan cleanup was performed using the paper disk method described in Merry et al. (2002) (Merry et al., 2002). Separation of 2-AB labeled glycans was performed by HPLC using a GlycoSep-N normal phase column according to Royle et al. (Royle, 2002) but modified to a three solvent system. Solvent A was 10 mM ammonium formate pH 4.4 in 80% acetonitrole. Solvent B was 30 mM ammonium formate pH 4.4. in 40% acetonitrile. Solvent C was 0.5% formic acid. The column temperature was 30° C. and 2-AB labeled glycans were detected by fluorescence (λex=330 nm, λem=420 nm). Gradient conditions were a linear gradient of 100% A to 100% B over 160 minutes at a flow rate of 0.4 ml/min, followed by 2 minutes 100% B to 100% C, increasing the flow rate to 1 ml/min. The column was washed for 5 minutes with 100% C, returning to 100% A over 2 minutes and running for 15 minutes at 100% A at a flow rate of 1 ml/min, then returning the flow rate to 0.4 ml/min for 5 minutes. All samples were injected in water.
The plasmids expressing the S. flexneri O-antigen with (SEQ ID NO: 29) or without (SEQ ID NO: 28) Z3206 were transformed into SCM3 cells (FIG. 10). Traces at late elution volumes shows a difference between the curves of the two samples containing the S. flexneri O antigen+/−Z3206 (FIG. 12). This difference in the elution pattern can be explained by a different oligosaccharide structure carrying a different monosaccharide at the reducing end: GlcNAc or GalNAc depending on the presence of the epimerase (Z3206).
Example 11 Analysis of pglB Specificity by Production and Characterization of Bioconjugate Produced from S. flexneri 6+/−Z3206 To assess whether pglB can transfer oligosaccharides having GlcNAc (S. flexneri 6 O-antigen) at the reducing end to the carrier protein EPA Nickel purified extracts from E. coli cells expressing EPA (SEQ ID NO: 25), PglB (SEQ ID NO: 26) and S. flexneri 6 O-antigen+/−Z3206 (SEQ ID NO: 29/SEQ ID NO: 28) were analyzed by western blot using anti EPA and anti type VI antibodies. The S. flexneri O6 antigen with and without GalNAc at the reducing end was transferred to EPA by PglB as detected by antiEPA and anti VI antisera (FIG. 13).
The O antigen is still produced and detected, but with lower production yield, which indicates that the efficiency of polysaccharide production without the epimerase is lower.
While this invention has been particularly shown and described with references to embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention encompassed by the claims. Such various changes that will be understood by those skilled in the art as covered within the scope of the invention include, in particular, N-glycosylated proteins and bioconjugates comprising a glycan other than those from E. coli and S. flexneri with GalNAc at the reducing terminus.
Sequence Listing
Applicant: GlycoVaxyn AG
Title: Biosynthetic System That Produces Immunogenic
Polysaccharides In Prokaryotic Cells
Number of SEQ ID NOs: 29
Nucleotide Sequence for E. coli O157 Z3206
Length: 993
Type: DNA
Organism: E. coli O157
Sequence:
SEQ ID NO: 1
ATGAACGATAACGTTTTGCTCATAGGAGCTTCCGGATTCGTAGGAACCCGACTACTTGAAACGG
CAATTGCTGACTTTAATATCAAGAAGCTGGACAAACAGCAGAGCCACTTTTATCCAGAAATCAC
ACAGATTGGCGATGTTCGCCATCAACAGGCACTGGACCAGGCGTTAGTCGGTTTTGACACTGTT
GTACTACTGGCAGCGGAACACCGCGATGACGTCAGCCCTACTTCTCTCTATTATGATGTCAACG
TTCAGGGTAGCCGCAATGTGCTGGCGGCCATGGAAAAAAATGGCGTTAAAAATATCATCTTTAC
CAGTTCCGTTGCTGTTTATGGTTTGAACAAACACAACCCTGACGAAAACCATCCACACGACCCT
TTGAACCACTACGGCAAAAGTAAGTGGCAGGCAGAGGAAGTGCTGCGTGAATGGTATAACAAAG
CACCAACAGAACGTTCATTAACCATCATCCGTGCTACCGTTATCTTCGGTGAACGCAACCGCGG
TAACGTCTATAACTTGCTGAAACAGATCGGTGGCGGCAAGTTTATGATGGTGGGCGCAGGGACT
AACTATAAGTCCATGGCTTATGTTGGAAACATTGTTGAGTTTATGAAGTACAAACTGAAGAATG
TTGCCGCAGGTTATGAGGTTTATAACTACGTTGATAAGCCAGACCTGAACATGAACCAGTTGGT
TGCTGAAGTTGAACAAAGCCTGAACAAAAAGATCCCTTCTATGCACTTGCCTTACCCACTAGGA
ATGCTGGGTGGATATTGCTTTGATATCCTGAGCAAAATTACGGGCAAAAAATACGCTGTCAGCT
CAGTGCGCGTGAAAAAATTCTGCGCAACAACACAGTTTGACGCAACGAAAGTGCATTCTTCAGG
TTTTGTGGCACCGTATACGCTGTCGCAAGGTCTGGATCGAAGACTGCAGTATGAATTCGTTCAT
GCCAAAAAAGACGACATAACGTTTGTTTCTGAG
Amino Acid Sequence for Z3206
Length: 331
Type: PRT
Organism: E coli O157
Sequence:
SEQ ID NO: 2
MNDNVLLIGASGFVGTRLLETAIADFNIKNLDKQQSHFYPEITQIGDVRDQQALDQALVGFDTV
VLLAAEHRDDVSPTSLYYDVNVQGTRNVLAAMEKNGVKNIIFTSSVAVYGLNKHNPDENHPHDP
FNHYGKSKWQAEEVLREWYNKAPTERSLTIIRPTVIFGERNRGNVYNLLKQIAGGKFMMVGAGT
NYKSMAYVGNIVEFIKYKLKNVAAGYEVYNYVDKPDLNMNQLVAEVEQSLNKKIPSMHLPYPLG
MLGGYCFDILSKITGKKYAVSSVRVKKFCATTQFDATKVHSSGFVAPYTLSQGLDRTLQYEFVH
AKKDDITFVSE
Nucleotide Sequence for E. coli O55 gne
Locus AF461121_1 BCT 2 May 2002
Definition (UDP-GlcNAc 4-epimerase Gne [Escherichia coil])
Accession AAL67550
Length: 993
Type: DNA
Organism: E. coli O55
Sequence:
SEQ ID NO: 3
ATGAACGATA ACGTTTTGCT CATAGGAGCT TCCGGATTCG TAGGAACCCG
ACTACTTGAA ACGGCAATTG CTGACTTTAA TATCAAGAAC CTGGACAAAC
AGCAGAGCCA CTTTTATCCA GAAATCACAC AGATTGGTGA TOTTCGTGAT
CAACAGGCAC TCGACCAGGC GTTAGCCGGT TTTGACACTG TTGTGCTACT
GGCAGCGGAA CACCGCGATG ACGTCAGCCC TACTTCTCTC TATTATGATG
TCAACGTTCA GGGTACCCGC AATGTGCTGG CGGCCATGGA AAAAAATGGC
GTTAAAAATA TCATCTTTAC CAGTTCCGTT GCTGTTTATG GTTTGAACAA
ACACAACCCT GACGAAAACC ATCCACACGA TCCTTTCAAC CACTACGGCA
AAAGTAAGTG GCAGGCAGAG GAAGTGCTGC GTGAATGGTA TAACAAAGCA
CCAACAGAAC GTTCATTAAC CATCATCCGT CCTACCGTTA TCTTCGGTGA
ACGGAACCGC GGTAACGTCT ATAACTTGCT GAAACAGATC GCTGGCGGCA
AGTTTATGAT GGTGGGCGCA GGGACTAACT ATAAGTCCAT GGCTTATGTT
GGAAACATTG TTGAGTTTAT CAAGTACAAA CTGAAGAATG TTGCCGCAGG
TTACGAGGTT TATAACTACG TTGATAAGCC AGACCTGAAC ATGAACCAGT
TGGTTGCTGA AGTTGAACAA AGCCTGAACA AAAAGATCCC TTCTATGCAC
TTGCCTTACC CACTAGGAAT GCTGGGTGGA TATTGCTTTG ATATCCTGAG
CAAAATTACG GGCAAAAAAT ACGCTGTCAG CTCTGTGCGC GTGAAAAAAT
TCTGCGCAAC AACACAGTTT GACGCAACGA NAGTGCATTC TTCAGGTTTT
GTGGCACCGT ATACGCTGTC GCAAGGTCTG GATCGAACTC TGCAGTATGA
ATTCGTCCAT GCCAAAAAAG ACGACATAAC GTTTGTTTCT GAG
Amino Acid Sequence for E. coli O55 UDP-GlcNAc 4-epimerase Gne
Locus AF461121_1
Definition (UDP-GlcNAc 4-epimerase Gne [Escherichia coli])
Accession AAL67550
Length: 331 aa linear
Type: PRT
Organism: E. coli O55
Sequence:
SEQ ID NO: 4
mndnvlliga sgfvgtrlle taiadfnikn ldkqqshfyp eitqigdvrd
qqaldqalag fdtvvllaae hrddvsptsl yydvnvqgtr nvlaamekng
vkniiftssv avyglnkhnp denhphdpfn hygkskwqae evirewynka
ptersltiir ptvifgernr gnvynllkqi aggkfmmvga gtnyksmayv
gnivefikyk lknvaagyev ynyvdkpdln mnqlvaeveq sinkkipsmh
lpyplgmlgg ycfdilskit gkkyayssvr vkkfcattqf datkvhssgf
vapytlsqgl drtlqyefvh akkdditfvs e
Nucleotide Sequence for E. coli O86 gne1
Locus AAO37706 BCT 6 Dec. 2005
Definition UDP-GlcNAc C4-epimerase [Escherichia coli O86].
Accession AAO37706
Length: 993
Type: DNA
Organism: E. coli O86
Sequence:
SEQ ID NO. 5
ATGAACGATA ACGTTTTGCT CATAGGAGCT TCCGGATTCG TAGGAACCCG
ACTACTTGAA ACGGCAATTG CTGACTTTAA TATCAAGAAC CTGGACAAAC
AGCAGAGCCA CTTTTATCCA GAAATCACAC AGATTGGTGA TGTTCGTGAT
CAACAGGCAC TCGACCAGGC GTTAGCCGGT TTTGACACTG TTGTACTACT
GGCAGCGGAA CACCGCGATG ACGTCAGCCC TACTTCTCTC TATTATGATG
TCAACGTTCA GGGTACCCGC AATGTGCTGG CGGCCATGGA AAAAAATGGC
GTTAAAAATA TCATCTTTAC CAGTTCCGTT GCTGTTTATG GTTTGAACAA
ACACAACCCT GACGAAAACC ATCCACACGA CCCTTTCAAC CACTACGGCA
AAAGCAAGTG GCAGGCGGAG GAAGTGCTGC GTGAATGGTA TAACAAAGCA
CCAACAGAAC GTTCATTAAC TATCATCCGT CCTACCGTTA TCTTCGGTGA
ACGCAACCGC GGTAACGTCT ATAACTTGCT GAAACAGATC GCTGGCGGCA
AGTTTATGAT GGTGGGCGCA GGGACTAACT ATAAGTCCAT GGCTTATGTT
GGAAACATTG TTGAGTTTAT CAAGTACAAA CTGAAGAATG TTGCCGCAGG
TTACGAGGTT TATAACTACG TTGATAAGCC AGACCTGAAC ATGAACCAGT
TGGTTGCTGA AGTTGAACAA AGCCTGAACA AAAAGATCCC TTCTATGCAC
TTGCCTTACC CACTAGGAAT GCTGGGTGGA TATTGCTTTG ATATCCTGAG
CAAAATTACG GGCAAAAAAT ACGCTGTCAG CTCTGTGCGC GTGAAAAAAT
TCTGCGCAAC AACACAGTTT GACGCAACGA AAGTGCATTC TTCAGGTTTT
GTGGCACCGT ATACGCTGTC GCAAGGTCTG GATCGAACTC TGCAGTATGA
ATTCGTCCAT GCCAAAAAAG ACGACATAAC GTTTGTTTCT GAG
Amino Acid Sequence for E. coli O86 UDP-GlcNAc C4-epimerase
Locus AA037706
Definition UDP-GlcNAc C4-epimerase [Escherichia coli O86].
Accession AAO37706
Length: 331 aa linear
Type: PRT
Organism: E. coli O86
Sequence:
SEQ ID NO: 6
mndnvlliga sgfvgtrlle taiadfnikn ldkqqshfyp eitqigdvrd
qqaldqalag fdtvvllaae hrddvsptsl yydvnvqgtr nvlaamekng
vkniiftssv avyglnkhnp denhphdpfn hygkskwqae evlrewynka
ptersltiir ptvifgernr gnvynllkqi aggkfmmvga gtnyksmayv
gnivefikyk lknvaagyev ynyvdkpdln mnqlvaeveq slnkkipsmh
lpyplgmlgg ycfdilskit gkkyayssvr vkkfcattqf datkvhssgf
vapytlsqgl drtlqyefvh akkdditfvs e
Nucleotide Sequence for Shigella boydii O18 gne
Locus ACD09753 BCT 5 May 2008
Definition UDP-N-acetylglucosamine 4-epimerase
[Shigella boydii CDC 3083-94].
Accession ACD09753
Length: 993
Type: DNA
Organism: Shigella boydii O18
Sequence:
SEQ ID NO: 7
ATGAACGATA ACGTTTTGCT CATAGGAGCT TCCGGATTCG TAGGAACCCG
ACTACTTGAA ACGGCAATTG CTGACTTTAA TATCAAGAAC CTGGACAAAC
AGCAGAGCCA TTTTTATCCA GCAATCACAC AGATTGGCGA TGTTCGTGAT
CAACAGGCAC TCGACCAGGC GTTAGCCGGT TTTGACACTG TTGTACTACT
GGCAGCGGAA CACCGCGATG ACGTCAGCCC TACTTCTCTC TATTATGATG
TCAACGTTCA GGGTACCCGC AATGTGCTGG CGGCCATGGA AAAAAATGGC
GTTAAAAATA TCATCTTTAC CAGTTCCGTT GCTGTTTATG GTTTGAACAA
ACACAACCCT GACGAAAACC ATCCACACGA CCCTTTCAAC CACTACGGCA
AAAGTAAGTG GCAGGCAGAG GAAGTGCTGC GTGAATGGTA TAACAAAGCA
CCAACAGAAC GTTCATTAAC CATCATCCGT CCTACCGTTA TCTTCGGTGA
ACGCAACCGC GGTAACGTCT ATAACTTGCT GAAACAGATC GCTGGCGGCA
AGTTTATGAT GGTGGGCGCA GGGACTAACT ATAAGTCCAT GGCTTATGTT
GGAAACATTG TTGAGTTTAT CAAGTACAAA CTGAAGAATG TTGCCGCAGG
TTATGAGGTT TATAACTATG TTGATAAGCC AGACCTGAAC ATGAACCAGT
TGGTTGCTGA AGTTGAACAA AGCCTGAACA AAAAGATCCC TTCTATGCAC
TTGCCTTACC CACTAGGAAT GCTGGGTGGA TATTGCTTTG ATATCCTGAG
CAAAATTACG GGCAAAAAAT ACGCTGTCAG CTCTGTGCGC GTGAAAAAAT
TCTGCGCAAC AACACAGTTT GACGCAACGA AAGTGCATTC TTCAGGTTTT
GTGGCACCGT ATACGCTGTC GCAAGGTCTG GATCGAACTC TGCAGTATGA
ATTCGTCCAT GCCAAAAAAG ACGACATAAC GTTTGTTTCT GAG
Amino Acid Sequence for Shigella boydii O18 UDP-N-
acetylglucosamine 4-epimerase
Locus ACD09753
Definition UDP-N-acetylglucosamine 4-epimerase
[Shigella boydii CDC 3083-94].
Accession ACD09753
Length: 331 aa linear
Type: PRT
Organism: Shigella boydii O18
Sequence:
SEQ ID NO: 8
mndnvlliga sgfvgtrile taiadfnikn ldkggshfyp aitqigdvrd
qqaldqalag fdtvvliaae hrddvsptsi yydvnvqgtr nvlaamekng
vkniiftssv avyglnkhnp denhphdpfn hygkskwqae evirewynka
ptersltiir ptvifgernr gnvynllkqi aggkfmmvga gtnyksmayv
gnivefikyk lknvaagyev ynyvdkpdln mnqlvaeveq sinkkipsmh
lpyplgmlgg ycfdilskit gkkyayssvr vkkfcattqf datkvhssgf
vapytlsggl drtlqyefvh akkdditfvs
Nucleotide Sequence for Salmonella enterica O30 gne
Locus AAV34516 BCT 25 Oct. 2004
Definition UDP-GlcNAc 4-epimerase
[Salmonella enterica subsp. salamae serovar Greenside].
Accession AAV34516
Length: 993
Type: DNA
Organism: Salmonella enterica O30
Sequence:
SEQ ID NO: 9
ATGAACGATA ACGTTTTGCT CATTGGTGCT TCCGGATTCG TAGGAACCCG
ACTCCTTGAA ACGGCAGTGG ATGATTTTAA TATCAAGAAC CTGGATAAAC
AGCAAAGCCA TTTCTACCCA GAGATTACAC ACATTGGCGA TGTTCGTGAC
CAACAAATCC TTGACCAGAC GTTGGTGGGT TTTGACACCG TAGTACTATT
GGCTGCGGAG CATCGTGATG ATGTTAGTCC TACCTCGCTT TATTATGATG
TCAACGTCCA GGGAACGCGT AATGTACTGG CGGCGATGGA AAAAAATGGT
GTAAAAAATA TCATTTTTAC CAGTTCCGTT GCAGTTTATG GACTCAACAA
GAAAAATCCT GACGAAACGC ACCCTCACGA TCCCTTTAAT CATTACGGAA
AAAGTAAATG GCAAGCAGAA GAAGTTCTGC GTGAGTGGCA TGCTAAAGCG
CCGAATGAGC GTTCTTTGAC CATAATTCGT CCTACCGTTA TTTTCGGGGA
GCGTAACCGC GGTAATGTAT ACAATCTCTT GAAACAGATC GCTGGTGGTA
AATTTGCGAT GGTTGGTCCG GGAACTAACT ATAAATCAAT GGCTTATGTT
GGTAATATCG TTGAGTTTAT CAAATTCAAA CTCAAGAATG TTACGGCGGG
CTATGAAGTT TATAATTATG TTGATAAACC TGATCTGAAT ATGAATCAAT
TGGTTGCTGA AGTAGAGCAG AGCCTGGGCA AAAAAATACC ATCGATGCAC
CTTCCATATC CATTAGGTAT GCTGGGGGGT TACTGTTTCG ATATCCTGAG
CAAAGTAACG GGCAAGAAGT ACGCTGTAAG TTCGGTTCGT GTTAAAAAAT
TCTGTGCGAC AACGCAGTTT GATGCAACAA AAGTGCATTC TTCTGGTTTT
GTTGCGCCAT ACACCTTATC TCAGGGGTTG GATCGTACAC TGCAATATGA
ATTTGTTCAT GCAAAGAAAG ATGACATTAC ATTCGTTTCA GAG
Amino Acid Sequence for Salmonella enterica O30 UDP-
GlcNAc 4-epimerase
Locus AAV34516
Definition UDP-GlcNAc 4-epimerase
[Salmonella enterica subsp. salamae serovar Greenside].
Accession AAV34516
Length: 331 aa linear
Type: PRT
Organism: Salmonella enterica O30
Sequence:
SEQ ID NO: 10
mndnviliga sgfvgtrlle tavddfnikn ldkggshfyp eithigdvrd
ggildgtivg fdtvvilaae hrddvsptsl yydvnvqgtr nvlaamekng
vkniiftssv avyglnkknp dethphdpfn hygkskwgae evlrewhaka
pnersltiir ptvifgernr gnvyralkgi aggkfamvgp gtnyksmayv
gnivefikfk lknvtagyev ynywdkpdln mnglvaeveg slgkkipsmh
lpyplgmlgg ycfdilskvt gkkyayssvr vkkfcattqf datkvhssgf
vapytlsggl drtlgyefvh akkdditfvs e
Nucleotide Sequence for C. jejuni gne
Locus YP_002344524 BCT 14 Sep. 2010
Definition UDP-GlcNAc/Glc 4-epimerase
[Campylobacter jejuni subsp. jejuni
Accession YP_002344524
Length: 987
Type: DNA
Organism: C. jejuni
Sequence:
SEQ ID NO: 11
ATGAAAATTCTTATTAGCGGTGGTGCAGGTTATATAGGTTCTCATACTTTAAGACAATT
TTTAAAAACAGATCATGAAATTTGTGTTTTAGATAATCTTTCTAAGGGTTCTAAAATCG
CAATAGAAGATTTGCAAAAAACAAGAGCTTTTAAATTTTTCGAACAAGATTTAAGTGAT
TTTCAAGGCGTAAAAGCATTGTTTGAGAGAGAAAAATTTGACGCTATTGTGCATTTTGC
AGCAAGCATTGAAGTTTTTGAAAGTATGCAAAATCCTTTAAAATATTATATGAACAACA
CTGTTAATACGACAAATCTCATCGAAACTTGTTTGCAAACTGGAGTGAATAAATTTATA
TTTTCTTCAACGGCGGCCACTTATGGCGAACCACAAACTCCCGTTGTGAGCGAAACAAG
TCCTTTAGCACCTATTAATCCTTATGGGCGTAGTAAGCTTATGAGTGAAGAAGTTTTGC
GTGATGCAAGTATGGCAAATCCTGAATTTAAGCATTGTATTTTAAGATATTTTAATGTT
GCAGGTGCTTGTATGGATTATACTTTAGGACAACGCTATCCAAAAGCGACTTTGCTTAT
AAAAGTTGCAGCTGAATGTGCCGCAGGAAAACGTGATAAACTTTTCATATTTGGCGATG
ATTATGATACAAAAGATGGTACTTGCATAAGAGATTTTATCCATGTAGATGATATTTCA
AGTGCACATTTAGCGGCTTTGGATTATTTAAAAGAGAATGAAAGCAATGTTTTTAATGT
AGGTTATGGACATGGTTTTAGCGTAAAAGAAGTGATTGAAGCGATGAAAAAAGTTAGCG
GAGTGGATTTTAAAGTAGAACTTGCCCCACGCCGTGCGGGTGATCCTAGTGTATTGATT
TCTGATGCAAGTAAAATCAGAAATCTTACTTCTTGGCAGCCTAAATATGATGATTTAGA
GCTTATTTGTAAATCTGCTTTTGATTGGGAAAAACAGTGTTAA
Amino Acid Sequence for C. jejuni UDP-GlcNAc/Glc 4-epimerase
Locus YP_002344524
Definition UDP-GlcNAc/Glc 4-epimerase
[Campylobacter jejuni subsp. jejuni
Accession YP_002344524
Length: 328 aa linear
Type: PRT
Organism: C. jejuni
Sequence:
SEQ ID NO: 12
mkilisggag yigshtlrqf lktdheicvl dnlskgskia iedlqktraf
kffeqdlsdf qgvkalfere kfdaivhfaa sievfesmqn plkyymnntv
nttnlietcl gtgvnkfifs staatygepq tpvvsetspl apinpygrsk
imseevirda smanpefkhc ilryfnvaga cmdytlaqry pkatllikva
aecaagkrdk ififgddydt kdgtcirdfi hvddissahi aaldylkene
snvfnvgygh gfsvkeviea mkkvsgvdfk velaprragd psvlisdask
irnltswqpk yddlelicks afdwekqc
Nucleotide Sequence for E. coli K12 galE
Locus AP_001390 BCT 30 Apr. 2010
Definition UDP-galactose-4-epimerase
[Escherichia coli str. K-12 substr. W3110].
Accession AP_001390
Length: 1,017
Type: DNA
Organism: E. coli K12
Sequence:
SEQ ID NO: 13
ATGAGAGTTCTGGTTACCGGTGGTAGCGGTTACATTGGAAGTCATACCTGTGTGCAA
TTACTGCAAAACGGTCATGATGTCATCATTCTTGATAACCTCTGTAACAGTAAGCGC
AGCGTACTGCCTGTTATCGAGCCTTTTAGGCGGCAAACATCCAACGTTTGTTGAAGG
CGATATTCGTAACGAAGCGTTGATGACCGAGATCCTGCACGATCACGCTATCGACAC
CGTGATCCACTTCGCCGGGCTGAAAGCCGTGGGCGAATCGGTACAAAAACCGCTGGA
ATATTACGACAACAATGTCAACGGCACTCTGCGCCTGATTAGCGCCATGCGCGCCGC
TAACGTCAAAAACTTTATTTTTAGCTCCTCCGCCACCGTTTATGGCGATCAGCCCAA
AATTCCATACGTTGAAAGCTTCCCGACCGGCACACCGCAAAGCCCTTACGGCAAAAG
CAAGCTGATGGTGGAACAGATCCTCACCGATCTGCAAAAAGCCCAGCCGGACTGGAG
CATTGCCCTGCTGCGCTACTTCAACCCGGTTGGCGCGCATCCGTCGGGCGATATGGG
CGAAGATCCGCAAGGCATTCCGAATAACCTGATGCCATACATCGCCCAGGTTGCTGT
AGGCCGTCGCGACTCGCTGGCGATTTTTGGTAACGATTATCCGACCGAAGATGGTAC
TGGCGTACGCGATTACATCCACGTAATGGATCTGGCGGACGGTCACGTCGTGGCGAT
GGAAAAACTGGCGAACAAGCCAGGCGTACACATCTACAACCTCGGCGCTGGCGTAGG
CAACAGCGTGCTGGACGTGGTTAATGCCTTCAGCAAAGCCTGCGGCAAACCGGTTAA
TTATCATTTTGCACCGCGTCGCGAGGGCGACCTTCCGGCCTACTGGGCGGACGCCAG
CAAAGCCGACCGTGAACTGAACTGGCGCGTAACGCGCACACTCGATGAAATGGCGCA
GGACACCTGGCACTGGCAGTCACGCCATCCACAGGGATATCCCGATTAA
Amino Acid Sequence for E. coli K12 UDP-galactose-4-epimerase
Locus AP_001390
Definition UDP-galactose-4-epimerase
[Escherichia coli str. K-12 substr. W3110].
Accession AP_001390
Length: 338 aa linear
Type: PRT
Organism: E. coli K12
Sequence:
SEQ ID NO: 14
mrvlvtqgsgyigshtcvqllqnghdviildnlcnskrsvlpvierlggkhptfvegdi
rnealmteilhdhaidtvihfaglkavgesvqkpleyydnnvngtlrlisamraanvkn
fifsssatvygdqpkipyvesfptgtpqspygksklmveqi1tdlqkaqpdwsiallry
fnpvgahpsgdmgedpqgipnnlmpyiaqvavgrrdslaifgndyptedgtgvrdyihv
mdladghvvameklankpgvhiynigagvgnsvldvvnafskacgkpvnyhfaprregd
lpaywadaskadrelnwrvtrtldemaqdtwhwqsrhpqgypd
Nucleotide Sequence for E. coli O86 gne2
Locus AAV85952 BCT 27 Mar. 2005
Definition Gne [Escherichia coli O86[.
Accession AAV85952
Length: 1,020
Type: DNA
Organism: E. coli O86
Sequence:
SEQ ID NO: 15
ATGGTGATTT TCGTAACAGG CGGTGCAGGA TATATTGGAT CCCATACCAT
ACTTGAGTTA CTTAATAATC GTCATGATGT CGTTTCGATA GATAATTTTG
TCAATTCCTC TATAGAATCA TTAAAAAGAC TAGAGCAAAT AACTAATAAG
AAAATTATTT CTTATCAAGG TGATATCCGT GATAAAAATC TACTTGATGA
GATTTTTTCA AGACACCATA TCCATGCTGT AATTCACTTT GCATCGTTAA
AATCTGTAGG TGAGTCTAAG TTAAAGCCCT TAGAGTATTA TTCTAATAAT
GTTGGTGGAA CTTTAGTATT ACTTCAATGC ATGAAGAGAT ATAACATTAA
TAAAATGATA TTTAGCTCTT CTGCTACTGT TTATGGGAGT AACAGTATCC
CTCCCCATAC GGAAGATAGA CGAATTGGTG AAACTACAAA CCCATATGGG
ACATCGAAAT TTATAATAGA AATAATTTTG AGTGATTATT GTGATAGTGA
TAATAATAAA TCAGTAATTG CACTGCGTTA CTTTAATCCA ATCGGAGCAC
ATAAGTCCGG GATGATTGGT GAAAATCCTA ACGGGATCCC TAATAATCTG
GTTCCTTATA TATCTAAAGT TGCACAAAAT CAACTTCCTG TATTAAATAT
TTATGGCAAC GATTATCCAA CTAAAGATGG TACAGGAGTA AGAGACTATA
TACATGTCTG TGATTTGGCT AAAGGGCATG TTAAAGCATT AGAATATATG
TTTTTAAATG ATGTCAATTA TGAAGCTTTT AATTTAGGTA CTGGTCAAGG
TTATTCTGTT TTAGAGATTG TAAAAATGTT TGAGATAGTC ACTAAAAAGA
GTATACCTGT TGCTATTTGT AATAGACGTG AGGGGGATGT TGCGGAGTCA
TGGGCGTCTG CTGATTTGGC ACATAAAAAG CTTTCCTGGA AAGCGCAAAA
AAATTTGAAA GAAATGATCG AAGATGTATG GCGTTGGCAA ACAAACAATC
CAAATGGATA TAAAAAATAA
Amino Acid Sequence for E. coli O86 Gne
Locus AAV85952
Definition Gne [Escherichia coli O86].
Accession AAV85952
Length: 339 aa (gne2) linear
Type: PRT
Organism: E. coli O86
Sequence:
SEQ ID NO: 16
mvifvtggag yigshtilel innghdvvsi dnfvnssies lkrvegitnk
kiisyggdir dknlldeifs rhhidavihf aslksvgesk lkpleyysnn
vgctivllec mkryninkmi fsssatvygs nsipphtedr rigettnpyg
tskfiieiil sdycdsdnnk svialryfnp igahksgmig enpngipnnl
vpyiskvaqn qlpviniygn dyptkdgtgv rdyihvcdla kghvkaleym
findvnyeaf nlgtgqgysv leivkmfeiv tkksipvaic nrregdvaes
wasadlahkk lswkaeknlk emiedvwrwq tnnpngykk
Nucleotide Sequence for synthetic oligonucleotide Z3206-
Fw (primer) encoding an end of Z3206; restriction sites underlined
Length: 30
Type: DNA
Sequence:
SEQ ID NO: 17
AAACCCGGGATGAACGATAACGTTTTGCTC
Nucleotide Sequence for synthetic oligonucleotide Z3206-
RvHA (primer) encoding an end of Z3206 with a hemoaglutinin
tag (HA tag); restriction sites underlined
Length: 60
Type: DNA
Organism:
Sequence:
SEQ ID NO: 18
AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTACTCAGAAACAAACGTTATGTC
Nucleotide Sequence for synthetic oligonucleotide gne-Fw
(primer) with restriction sites underlined
Length: 29
Type: DNA
Organism:
Sequence:
SEQ ID NO: 19
AAACCATGGATGAAAATTCTTATTAGCGG
Nucleotide Sequence for synthetic oligonucleotide gne-RV
(primer) with restriction sites underlined
Length: 57
Type: DNA
Organism:
Sequence:
SEQ ID NO: 20
AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTAGCACTGTTTTTCCCAATC
Nucleotide Sequence for oligonucleotide containing
restriction sites for NheI restriction enzyme
Length: 11
Type: DNA
Organism:
Sequence:
SEQ ID NO: 21
AAAAAGCTAGC
Nucleotide Sequence for oligonucleotide containing
restriction sites for AscI restriction enzyme
Length: 8
Type: DNA
Organism:
Sequence:
SEQ ID NO: 22
CCGCGCGG
Nucleotide Sequence for plasmid pMLBAD: Z3206 (E. coli O157
insert in plasmid) encoding Z3206 with a C-terminal hemagglutinin tag
Definition Ligation of product into Z3206-pMLBAD*
Features Location/Qualifiers
CDS 2105..3098
/label=Z3206
CDS 3098..3127
/label=HA
Length: 7794 bp
Type: DNA circular UNA
Sequence:
SEQ ID NO: 23
1 TCTACGGGGT CTGACGCTCA GTGGAACGAA ATCGATGAGC TCGCACGAAC CCAGTTGACA
61 TAAGCCTGTT CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC
121 AGAACCTTGA CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT
181 GACTGTTTTT TTGTACAGTC TAGCCTCGGG CATCCAAGCT AGCTAAGCGC GTTACGCCGT
241 GGGTCGATGT TTGATGTTAT GGAACAGCAA CGATGTTACG CAGCAGGGTA GTCGCCCTAA
301 AACAAAGTTA GGCAGCCGTT GTGCTGGTGC TTTCTAGTAG TTGTTGTGGG GTAGGCAGTC
361 AGAGCTCGAT TTGCTTGTCG CCATAATAGA TTCACAAGAA GGATTCGACA TGGGTCAAAG
421 TAGCGATGAA GCCAACGCTC CCGTTGCAGG GCAGTTTGCG CTTCCCCTGA GTGCCACCTT
481 TGGCTTAGGG GATCGCGTAC GCAAGAAATC TGGTGCCGCT TGGCAGGGTC AAGTCGTCGG
541 TTGGTATTGC ACAAAACTCA CTCCTGAAGG CTATGCGGTC GAGTCCGAAT CCCACCCAGG
601 CTCAGTGCAA ATTTATCCTG TGGCTGCACT TGAACGTGTG GCCTAAGCGA TATCTTAGGA
661 TCTCCCATCG GTGATGTCGG CGATATAGGC GCCAGCAACC GCACCTGTGG CGCCGGTGAT
721 GCCGGCCACG ATGCGTCCGG CGTAGAGGAT CTGCTCATGT TTGACAGCTT ATCATCGATG
781 CATAATGTGC CTGTCAAATG GACGAAGCAG GGATTCTGCA AACCCTATGC TACTCCGTCA
841 AGCCGTCAAT TGTCTGAATC GTTACCAATT ATGACAACTT GACGGCTACA TCATTCACTT
901 TTTCTTCACA ACCGGCACGG AACTCGCTCG GGCTGGCCCC GGTGCATTTT TTAAATACCC
961 GCGAGAAATA GAGTTGATCG TCAAAACCAA CATTGCGACC GACGGTGGCG ATAGGCATCC
1021 GGGTGGTGCT CAAAAGCAGC TTCGCCTGGC TGATACGTTG GTCCTCGCGC CAGCTTAAGA
1081 CGCTAATCCC TAACTGCTGG CGGAAAAGAT GTGACAGACG CGACGGCGAC AAGCAAACAT
1141 GCTGTGCGAC GCTGGCGATA TCAAAATTGC TGTCTGCCAG GTGATCGCTG ATGTACTGAC
1201 AAGCCTCGCG TACCCGATTA TCCATCGGTG GATGGAGCGA CTCGTTAATC GCTTCCATGC
1261 GCCGCAGTAA CAATTGCTCA AGCAGATTTA TCGCCAGCAG CTCCGAATAG CGCCCTTCCC
1321 CTTGCCCGGC GTTAATGATT TGCCCAAACA GGTCGCTGAA ATGCGGCTGG TGCGCTTCAT
1381 CCGGGCGAAA GAACCCCGTA TTGGCAAATA TTGACGGCCA GTTAAGCCAT TCATGCCAGT
1441 AGGCGCGCGG ACGAAAGTAA ACCCACTGGT GATACCATTC GCGAGCCTCC GGATGACGAC
1501 CGTAGTGATG AATCTCTCCT GGCGGGAACA GCAAAATATC ACCCGGTCGG CAAACAAATT
1561 CTCGTCCCTG ATTTTTCACC ACCCCCTGAC CGCGAATGGT GAGATTGAGA ATATAACCTT
1621 TCATTCCCAG CGGTCGGTCG ATAAAAAAAT CGAGATAACC GTTGGCCTCA ATCGGCGTTA
1681 AACCCGCCAC CAGATGGGCA TTAAACGAGT ATCCCGGCAG CAGGGGATCA TTTTGCGCTT
1741 CAGCCATACT TTTCATACTC CCGCCATTCA GAGAAGAAAC CAATTGTCCA TATTGCATCA
1301 GACATTGCCG TCACTGCGTC TTTTACTGGC TCTTCTCGCT AACCAAACCG GTAACCCCGC
1861 TTATTAAAAG CATTCTGTAA CAAAGCGGGA CCAAAGCCAT GACAAAAACG CGTAACAAAA
1921 GTGTCTATAA TCACGGCAGA AAAGTCCACA TTGATTATTT GCACGGCGTC ACACTTTGCT
1981 ATGCCATAGC ATTTTTATCC ATAAGATTAG CGGATCCTAC CTGACGCTTT TTATCGCAAC
2041 TCTCTACTGT TTCTCCATAC CCGTTTTTTT GGGCTAGCAG GAGGAATTCA CCATGGTACC
2101 CGGGATGAAC GATAACGTTT TGCTCATAGG AGCTTCCGGA TTCGTAGGAA CCCGACTACT
2161 TGAAACGGCA ATTGCTGACT TTAATATCAA GAACCTGGAC AAACAGCAGA GCCACTTTTA
2221 TCCAGAAATC ACACAGATTG GCGATGTTCG CGATCAACAG GCACTCGACC AGGCGTTAGT
2281 CGGTTTTGAC ACTGTTGTAC TACTGGCAGC GGAACACCGC GATGACGTCA GCCCTACTTC
2341 TCTCTATTAT GATGTCAACG TTCAGGGTAC CCGCAATGTG CTGGCGGCCA TGGAAAAAAA
2401 TGGCGTTAAA AATATCATCT TTACCAGTTC CGTTGCTGTT TATGGTTTGA ACAAACACAA
2461 CCCTGACGAA AACCATCCAC ACGACCCTTT CAACCACTAC GGCAAAAGTA AGTGGCAGGC
2521 AGAGGAAGTG CTGCGTGAAT GGTATAACAA AGCACCAACA GAACGTTCAT TAACCATCAT
2581 CCGTCCTACC GTTATCTTCG GTGAACGCAA CCGCGGTAAC GTCTATAACT TGCTGAAACA
2641 GATCGCTGGC GGCAAGTTTA TGATGGTGGG CGCAGGGACT AACTATAAGT CCATGGCTTA
2701 TGTTGGAAAC ATTGTTGAGT TTATCAAGTA CAAACTGAAG AATGTTGCCG CAGGTTATGA
2761 GGTTTATAAC TACGTTGATA AGCCAGACCT GAACATGAAC CAGTTGGTTG CTGAAGTTGA
2821 ACAAAGCCTG AACAAAAAGA TCCCTTCTAT GCACTTGCCT TACCCACTAG GAATGCTGGG
2881 TGGATATTGC TTTGATATCC TGAGCAAAAT TACGGGCAAA AAATACGCTG TCAGCTCAGT
2941 GCGCGTGAAA AAATTCTGCG CAACAACACA GTTTGACGCA ACGAAAGTGC ATTCTTCAGG
3001 TTTTGTGGCA CCGTATACGC TGTCGCAAGG TCTGGATCGA ACACTGCAGT ATGAATTCGT
3061 TCATGCCAAA AAAGACGACA TAACGTTTGT TTCTGAGTAC CCATACGATG TTCCAGATTA
3121 CGCTTAATCT AGAGTCGACC TGCAGGCATG CAAGCTTGGC TGTTTTGGCG GATGAGAGAA
3181 GATTTTCAGC CTGATACAGA TTAAATCAGA ACGCAGAAGC GGTCTGATAA AACAGAATTT
3241 GCCTGGCGGC AGTAGCGCGG TGGTCCCACC TGACCCCATG CCGAACTCAG AAGTGAAACG
3301 CCGTAGCGCC GATGGTAGTG TGGGGTCTCC CCATGCGAGA GTAGGGAACT GCCAGGCATC
3361 AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT TGTTTGTCGG
3421 TGAACGCTCT CCTGAGTAGG ACAAATCCGC CGGGAGCGGA TTTGAACGTT GCGAAGCAAC
3481 GGCCCGGAGG GTGGCGGGCA GGACGCCCGC CATAAACTGC CAGGCATCAA ATTAAGCAGA
3541 AGGCCATCCT GACGGATGGC CTTTTTGCGT TTCTACAAAC TCTTCCACTC ACTACAGCAG
3601 AGCCATTTAA ACAACATCCC CTCCCCCTTT CCACCGCGTC AGACGCCCGT AGCAGCCCGC
3661 TACGGGCTTT TTCATGCCCT GCCCTAGCGT CCAAGCCTCA CGGCCGCGCT CGGCCTCTCT
3721 GGCGGCCTTC TGGCGCTGAG GTCTGCCTCG TGAAGAAGGT GTTGCTGACT CATACCAGGC
3781 CTGAATCGCC CCATCATCCA GCCAGAAAGT GAGGGAGCCA CGGTTGATGA GAGCTTTGTT
3841 GTAGGTGGAC CAGTTGGTGA TTTTGAACTT TTGCTTTGCC ACGGAACGGT CTGCGTTGTC
3901 GGGAAGATGC GTGATCTGAT CCTTCAACTC AGCAAAAGTT CGATTTATTC AACAAAGCCG
3961 CCGTCCCGTC AAGTCAGCGT AATGCTCTGC CAGTGTTACA ACCAATTAAC CAATTCTGAT
4021 TAGAAAAACT CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA
4081 CCATATTTTT GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT
4141 AGGATGGCAA GATCCTGGTA TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT
4201 ATTAATTTCC CCTCGTCAAA AATAAGGTTA TCAAGCGAGA AATCACCATG AGTGACGACT
4261 GAATCCGGTG AGAATGGCAA AAGCTAAAAA GGCCGTAATA TCCAGCTGAA CGGTCTGGTT
4321 ATAGGTACAT TGAGCAACTG ACTGAAATGC CTCAAAATGT TCTTTACGAT GCCATTGGGA
4381 TATATCAACG GTGGTATATC CAGTGATTTT TTTCTCCATT TTAGCTTCCT TAGCTCCTGA
4441 AAATCTCGAT AACTCAAAAA ATACGCCCGG TAGTGATCTT ATTTCATTAT GGTGAAAGTT
4501 GGAACCTCTT ACGTGCCGAT CAACGTCTCA TTTTCGCCAA AAGTTGGCCC AGGGCTTCCC
4561 GGTATCAACA GGGACACCAG GATTTATTTA TTCTGCGAAG TGATCTTCCG TCACAGGTAT
4621 TTATTCGAAG ACGAAAGGGC CTCGTGATAC GCCTATTTTT ATAGGTTAAT GTCATGATAA
4681 TAATGGTTTC TTAGACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGCC CGCGTTCCTG
4741 CTGGCGCTGG GCCTGTTTCT GGCGCTGGAC TTCCCGCTGT TCCGTCAGCA GCTTTTCGCC
4801 CACGGCCTTG ATGATCGCGG CGGCCTTGGC CTGCATATCC CGATTCAACG GCCCCAGGGC
4861 GTCCAGAACG GGCTTCAGGC GCTCCCGAAG GTCTCGGGCC GTCTCTTGGG CTTGATCGGC
4921 CTTCTTGCGC ATCTCACGCG CTCCTGCGGC GGCCTGTAGG GCAGGCTCAT ACCCCTGCCG
4981 AACCGCTTTT GTCAGCCGGT CGGCCACGGC TTCCGGCGTC TCAACGCGCT TTGAGATTCC
5041 CAGCTTTTCG GCCAATCCCT GCGGTGCATA GGCGCGTGGC TCGACCGCTT GCGGGCTGAT
5101 GGTGACGTGG CCCACTGGTG GCCGCTCCAG GGCCTCGTAG AACGCCTGAA TGCGCGTGTG
5161 ACGTGCCTTG CTGCCCTCGA TGCCCCGTTG CAGCCCTAGA TCGGCCACAG CGGCCGCAAA
5221 CGTGGTCTGG TCGCGGGTCA TCTGCGCTTT GTTGCCGATG AACTCCTTGG CCGACAGCCT
5281 GCCGTCCTGC GTCAGCGGCA CCACGAACGC GGTCATGTGC GGGCTGGTTT CGTCACGGTG
5341 GATGCTGGCC GTCACGATGC GATCCGCCCC GTACTTGTCC GCCAGCCACT TGTGCGCCTT
5401 CTCGAAGAAC GCCGCCTGCT GTTCTTGGCT GGCCGACTTC CACCATTCCG GGCTGGCCGT
5461 CATGACGTAC TCGACCGCCA ACACAGCGTC CTTGCGCCGC TTCTCTGGCA GCAACTCGCG
5521 CAGTCGGCCC ATCGCTTCAT CGGTGCTGCT GGCCGCCCAG TGCTCGTTCT CTGGCGTCCT
5581 GCTGGCGTCA GCGTTGGGCG TCTCGCGCTC GCGGTAGGCG TGCTTGAGAC TGGCCGCCAC
5641 GTTGCCCATT TTCGCCAGCT TCTTGCATCG CATGATCGCG TATGCCGCCA TGCCTGCCCC
5701 TCCCTTTTGG TGTCCAACCG GCTCGACGGG GGCAGCGCAA GGCGGTGCCT CCGGCGGGCC
5761 ACTCAATGCT TGAGTATACT CACTAGACTT TGCTTCGCAA AGTCGTGACC GCCTACGGCG
5821 GCTGCGGCGC CCTACGGGCT TGCTCTCCGG GCTTCGCCCT GCGCGGTCGC TGCGCTCCCT
5881 TGCCAGCCCG TGGATATGTG GACGATGGCC GCGAGCGGCC ACCGGCTGGC TCGCTTCGCT
5941 CGGCCCGTGG ACAACCCTGC TGGACAAGCT GATGGACAGG CTGCGCCTGC CCACGAGCTT
6001 GACCACAGGG ATTGCCCACC GGCTACCCAG CCTTCGACCA CATACCCACC GGCTCCAACT
6061 GCGCGGCCTG CGGCCTTGCC CCATCAATTT TTTTAATTTT CTCTGGGGAA AAGCCTCCGG
6121 CCTGCGGCCT GCGCGCTTCG CTTGCCGGTT GGACACCAAG TGGAAGGCGG GTCAAGGCTC
6181 GCGCAGCGAC CGCGCAGCGG CTTGGCCTTG ACGCGCCTGG AACGACCCAA GCCTATGCGA
6241 GTGGGGGCAG TCGAAGGCGA AGCCCGCCCG CCTGCCCCCC GAGCCTCACG GCGGCGAGTG
6301 CGGGGGTTCC AAGGGGGCAG CGCCACCTTG GGCAAGGCCG AAGGCCGCGC AGTCGATCAA
6361 CAAGCCCCGG AGGGGCCACT TTTTGCCGGA GGGGGAGCCG CGCCGAAGGC GTGGGGGAAC
6421 CCCGCAGGGG TGCCCTTCTT TGGGCACCAA AGAACTAGAT ATAGGGCGAA ATGCGAAAGA
6481 CTTAAAAATC AACAACTTAA AAAAGGGGGG TACGCAACAG CTCATTGCGG CACCCCCCGC
6541 AATAGCTCAT TGCGTAGGTT AAAGAAAATC TGTAATTGAC TGCCACTTTT ACGCAACGCA
6601 TAATTGTTGT CGCGCTGCCG AAAAGTTGCA GCTGATTGCG CATGGTGCCG CAACCGTGCG
6661 GCACCCTACC GCATGGAGAT AAGCATGGCC ACGCAGTCCA GAGAAATCGG CATTCAAGCC
6721 AAGAACAAGC CCGGTCACTG GGTGCAAACG GAACGCAAAG CGCATGAGGC GTGGGCCGGG
6781 CTTATTGCGA GGAAACCCAC GGCGGCAATG CTGCTGCATC ACCTCGTGGC GCAGATGGGC
6841 CACCAGAACG CCGTGGTGGT CAGCCAGAAG ACACTTTCCA AGCTCATCGG ACGTTCTTTG
6901 CGGACGGTCC AATACGCAGT CAAGGACTTG GTGGCCGAGC GCTGGATCTC CGTCGTGAAG
6961 CTCAACGGCC CCGGCACCGT GTCGGCCTAC GTGGTCAATG ACCGCGTGGC GTGGGGCCAG
7021 CCCCGCGACC AGTTGCGCCT GTCGGTGTTC AGTGCCGCCG TGGTGGTTGA TCACGACGAC
7081 CAGGACGAAT CGCTGTTGGG GCATGGCGAC CTGCGCCGCA TCCCGACCCT GTATCCGGGC
7141 GAGCAGCAAC TACCGACCGG CCCCGGCGAG GAGCCGCCCA GCCAGCCCGG CATTCCGGGC
7201 ATGGAACCAG ACCTGCCAGC CTTGACCGAA ACGGAGGAAT GGGAACGGCG CGGGCAGCAG
7261 CGCCTGCCGA TGCCCGATGA GCCGTGTTTT CTGGACGATG GCGAGCCGTT GGAGCCGCCG
7321 ACACGGGTCA CGCTGCCGCG CCGGTAGCAC TTGGGTTGCG CAGCAACCCG TAAGTGCGCT
7381 GTTCCAGACT ATCGGCTGTA GCCGCCTCGC CGCCCTATAC CTTGTCTGCC TCCCCGCGTT
7441 GCGTCGCGGT GCATGGAGCC GGGCCACCTC GACCTGAATG GAAGCCGGCG GCACCTCGCT
7501 AACGGATTCA CCGTTTTTAT CAGGCTCTGG GAGGCAGAAT AAATGATCAT ATCGTCAATT
7561 ATTACCTCCA CGGGGAGAGC CTGAGCAAAC TGGCCTCAGG CATTTGAGAA GCACACGGTC
7621 ACACTGCTTC CGGTAGTCAA TAAACCGGTA AACCAGCAAT AGACATAAGC GGCTATTTAA
7681 CGACCCTGCC CTGAACCGAC GACCGGGTCG AATTTGCTTT CGAATTTCTG CCATTCATCC
7741 GCTTATTATC ACTTATTCAG GCGTAGCACC AGGCGTTTAA GTCGACCAAT AACC
Nucleotide Sequence for pMLBAD: gne (E. coli O157 insert
in plasmid) which encodes Gne with a C-terminal hemagglutinin tag
Locus gne-pMLBAD
Definition Ligation of dig galE into pmlbad did (NcoI-XbaI)
Features Location/Qualifiers
CDS 2097..3080
/label=galE
CDS 3081..3107
/label=HA
Region 3108..3110
/label=stop
Length: 7776 bp
Type: DNA circular UNA
Sequence:
SEQ ID NO: 24
1 TCTACGGGGT CTGACGCTCA GTGGAACGAA ATCGATGAGC TCGCACGAAC CCAGTTGACA
61 TAAGCCTGTT CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC
121 AGAACCTTGA CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT
181 GACTGTTTTT TTGTACAGTC TAGCCTCGGG CATCCAAGCT AGCTAAGCGC GTTACGCCGT
241 GGGTCGATGT TTGATGTTAT GGAACAGCAA CGATGTTACG CAGCAGGGTA GTCGCCCTAA
301 AACAAAGTTA GGCAGCCGTT GTGCTGGTGC TTTCTAGTAG TTGTTGTGGG GTAGGCAGTC
361 AGAGCTCGAT TTGCTTGTCG CCATAATAGA TTCACAAGAA GGATTCGACA TGGGTCAAAG
421 TAGCGATGAA GCCAACGCTC CCGTTGCAGG GCAGTTTGCG CTTCCCCTGA GTGCCACCTT
481 TGGCTTAGGG GATCGCGTAC GCAAGAAATC TGGTGCCGCT TGGCAGGGTC AAGTCGTCGG
541 TTGGTATTGC ACAAAACTCA CTCCTGAAGG CTATGCGGTC GAGTCCGAAT CCCACCCAGG
601 CTCAGTGCAA ATTTATCCTG TGGCTGCACT TGAACGTGTG GCCTAAGCGA TATCTTAGGA
661 TCTCCCATCG GTGATGTCGG CGATATAGGC GCCAGCAACC GCACCTGTGG CGCCGGTGAT
721 GCCGGCCACG ATGCGTCCGG CGTAGAGGAT CTGCTCATGT TTGACAGCTT ATCATCGATG
781 CATAATGTGC CTGTCAAATG GACGAAGCAG GGATTCTGCA AACCCTATGC TACTCCGTCA
841 AGCCGTCAAT TGTCTGATTC GTTACCAATT ATGACAACTT GACGGCTACA TCATTCACTT
901 TTTCTTCACA ACCGGCACGG AACTCGCTCG GGCTGGCCCC GGTGCATTTT TTAAATACCC
961 GCGAGAAATA GAGTTGATCG TCAAAACCAA CATTGCGACC GACGGTGGCG ATAGGCATCC
1021 GGGTGGTGCT CAAAAGCAGC TTCGCCTGGC TGATACGTTG GTCCTCGCGC CAGCTTAAGA
1081 CGCTAATCCC TAACTGCTGG CGGAAAAGAT GTGACAGACG CGACGGCGAC AAGCAAACAT
1141 GCTGTGCGAC GCTGGCGATA TCAAAATTGC TGTCTGCCAG GTGATCGCTG ATGTACTGAC
1201 AAGCCTCGCG TACCCGATTA TCCATCGGTG GATGGAGCGA CTCGTTAATC GCTTCCATGC
1261 GCCGCAGTAA CAATTGCTCA AGCAGATTTA TCGCCAGCAG CTCCGAATAG CGCCCTTCCC
1321 CTTGCCCGGC GTTAATGATT TGCCCAAACA GGTCGCTGAA ATGCGGCTGG TGCGCTTCAT
1381 CCGGGCGAAA GAACCCCGTA TTGGCAAATA TTGACGGCCA GTTAAGCCAT TCATGCCAGT
1441 AGGCGCGCGG ACGAAAGTAA ACCCACTGGT GATACCATTC GCGAGCCTCC GGATGACGAC
1501 CGTAGTGATG AATCTCTCCT GGCGGGAACA GCAAAATATC ACCCGGTCGG CAAACAAATT
1561 CTCGTCCCTG ATTTTTCACC ACCCCCTGAC CGCGAATGGT GAGATTGAGA ATATAACCTT
1621 TCATTCCCAG CGGTCGGTCG ATAAAAAAAT CGAGATAACC CTTGGCCTCA ATCGGCGTTA
1681 AACCCGCCAC CAGATGGGCA TTAAACGAGT ATCCCGGCAG CAGGGGATCA TTTTGCGCTT
1741 CAGCCATACT TTTCATACTC CCGCCATTCA GAGAAGAAAC CAATTGTCCA TATTGCATCA
1801 GACATTGCCG TCACTGCGTC TTTTACTGGC TCTTCTCGCT AACCAAACCG GTAACCCCGC
1861 TTATTAAAAG CATTCTGTAA CAAAGCGGGA CCAAAGCCAT GACAAAAACG CGTAACAAAA
1921 GTGTCTATAA TCACGGCAGA AAAGTCCACA TTGATTATTT GCACGGCGTC ACACTTTGCT
1981 ATGCCATAGC ATTTTTATCC ATAAGATTAG CGGATCCTAC CTGACGCTTT TTATCGCAAC
2041 TCTCTACTGT TTCTCCATAC CCGTTTTTTT GGGCTAGCAG GAGGAATTCA CCATGGATGA
2101 AAATTCTTAT TAGCGGTGGT GCAGGTTATA TAGGTTCTCA TACTTTAAGA CAATTTTTAA
2161 AAACAGATCA TGAAATTTGT GTTTTAGATA ATCTTTCTAA GGGTTCTAAA ATCGCAATAG
2221 AAGATTTGCA AAAAATAAGA ACTTTTAAAT TTTTTGAACA AGATTTAAGT GATTTTCAAG
2281 GCGTAAAAGC ATTGTTTGAG AGAGAAAAAT TTGACGCTAT TGTGCATTTT GCAGCGAGCA
2341 TTGAAGTTTT TGAAAGTATG CAAAACCCTT TAAAGTATTA TATGAATAAC ACTGTTAATA
2401 CGACAAATCT CATCGAAACT TGTTTGCAAA CTGGAGTGAA TAAATTTATA TTTTCTTCAA
2461 CGGCAGCCAC TTATGGCGAA CCACAAACTC CCGTTGTGAG CGAAACAAGT CCTTTAGCAC
2521 CTATTAATCC TTATGGGCGT AGTAAGCTTA TGAGCGAAGA GGTTTTGCGT GATGCAAGTA
2581 TGGCAAATCC TGAATTTAAG CATTGTATTT TAAGATATTT TAATGTTGCA GGTGCTTGCA
2641 TGGATTATAC TTTAGGACAA CGCTATCCAA AAGCGACTTT GCTTATAAAA GTTGCAGCTG
2701 AATGTGCCGC AGAAAAACGT AATAAACTTT TCATATTTGG CGATGATTAT GATACAAAAG
2761 ATGGCACTTG CATAAGAGAT TTTATCCATG TGGATGATAT TTCAAGTGCG CATTTATCGG
2821 CTTTGGATTA TTTAAAAGAG AATGAAAGCA ATGTTTTTAA TGTAGGTTAT GGACATGGTT
2881 TTAGCGTAAA AGAAGTGATT GAAGCGATGA AAAAAGTTAG CGGAGTGGAT TTTAAAGTAG
2941 AACTTGCCCC ACGCCGTGCG GGTGATCCTA GTGTATTGAT TTCTGATGCA AGTAAAATCA
3001 GAAATCTTAC TTCTTGGCAG CCTAAATATG ATGATTTAGG GCTTATTTGT AAATCTGCTT
3061 TTGATTGGGA AAAACAGTGC TACCCATACG ATGTTCCAGA TTACGCTTAA TCTAGAGTCG
3121 ACCTGCAGGC ATGCAAGCTT GGCTGTTTTG GCGGATGAGA GAAGATTTTC AGCCTGATAC
3181 AGATTAAATC AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG
3241 CGGTGGTCCC ACCTGACCCC ATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA
3301 GTGTGGGGTC TCCCCATGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT
3361 CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGTGAACGC TCTCCTGAGT
3421 AGGACAAATC CGCCGGGAGC GGATTTGAAC GTTGCGAAGC AACGGCCCGG AGGGTGGCGG
3481 GCAGGACGCC CGCCATAAAC TGCCAGGCAT CAAATTAAGC AGAAGGCCAT CCTGACGGAT
3541 GGCCTTTTTG CGTTTCTACA AACTCTTCCA CTCACTACAG CAGAGCCATT TAAACAACAT
3601 CCCCTCCCCC TTTCCACCGC GTCAGACGCC CGTAGCAGCC CGCTACGGGC TTTTTCATGC
3661 CCTGCCCTAG CGTCCAAGCC TCACGGCCGC GCTCGGCCTC TCTGGCGGCC TTCTGGCGCT
3721 GAGGTCTGCC TCGTGAAGAA GGTGTTGCTG ACTCATACCA GGCCTGAATC GCCCCATCAT
3781 CCAGCCAGAA AGTGAGGGAG CCACGGTTGA TGAGAGCTTT GTTGTAGGTG GACCAGTTGG
3841 TGATTTTGAA CTTTTGCTTT GCCACGGAAC GGTCTGCGTT GTCGGGAAGA TGCGTGATCT
3901 GATCCTTCAA CTCAGCAAAA GTTCGATTTA TTCAACAAAG CCGCCGTCCC GTCAAGTCAG
3961 CGTAATGCTC TGCCAGTGTT ACAACCAATT AACCAATTCT GATTAGAAAA ACTCATCGAG
4021 CATCAAATGA AACTGCAATT TATTCATATC AGGATTATCA ATACCATATT TTTGAAAAAG
4081 CCGTTTCTGT AATGAAGGAG AAAACTCACC GAGGCAGTTC CATAGGATGG CAAGATCCTG
4141 GTATCGGTCT GCGATTCCGA CTCGTCCAAC ATCAATACAA CCTATTAATT TCCCCTCGTC
4201 AAAAATAAGG TTATCAAGCG AGAAATCACC ATGAGTGACG ACTGAATCCG GTGAGAATGG
4261 CAAAAGCTAA AAAGGCCGTA ATATCCAGCT GAACGGTCTG GTTATAGGTA CATTGAGCAA
4321 CTGACTGAAA TGCCTCAAAA TGTTCTTTAC GATGCCATTG GGATATATCA ACGGTGGTAT
4381 ATCCAGTGAT TTTTTTCTCC ATTTTAGCTT CCTTAGCTCC TGAAAATCTC GATAACTCAA
4441 AAAATACGCC CGGTAGTGAT CTTATTTCAT TATGGTGAAA GTTGGAACCT CTTACGTGCC
4501 GATCAACGTC TCATTTTCGC CAAAAGTTGG CCCAGGGCTT CCCGGTATCA ACAGGGACAC
4561 CAGGATTTAT TTATTCTGCG AAGTGATCTT CCGTCACAGG TATTTATTCG AAGACGAAAG
4621 GGCCTCGTGA TACGCCTATT TTTATAGGTT AATGTCATGA TAATAATGGT TTCTTAGACG
4681 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GCCCGCGTTC CTGCTGGCGC TGGGCCTGTT
4741 TCTGGCGCTG GACTTCCCGC TGTTCCGTCA GCAGCTTTTC GCCCACGGCC TTGATGATCG
4801 CGGCGGCCTT GGCCTGCATA TCCCGATTCA ACGGCCCCAG GGCGTCCAGA ACGGGCTTCA
4861 GGCGCTCCCG AAGGTCTCGG GCCGTCTCTT GGGCTTGATC GGCCTTCTTG CGCATCTCAC
4921 GCGCTCCTGC GGCGGCCTGT AGGGCAGGCT CATACCCCTG CCGAACCGCT TTTGTCAGCC
4981 GGTCGGCCAC GGCTTCCGGC GTCTCAACGC GCTTTGAGAT TCCCAGCTTT TCGGCCAATC
5041 CCTGCGGTGC ATAGGCGCGT GGCTCGACCG CTTGCGGGCT GATGGTGACG TGGCCCACTG
5101 GTGGCCGCTC CAGGGCCTCG TAGAACGCCT GAATGCGCGT GTGACGTGCC TTGCTGCCCT
5161 CGATGCCCCG TTGCAGCCCT AGATCGGCCA CAGCGGCCGC AAACGTGGTC TGGTCGCGGG
5221 TCATCTGCGC TTTGTTGCCG ATGAACTCCT TGGCCGACAG CCTGCCGTCC TGCGTCAGCG
5281 GCACCACGAA CGCGGTCATG TGCGGGCTGG TTTCGTCACG GTGGATGCTG GCCGTCACGA
5341 TGCGATCCGC CCCGTACTTG TCCGCCAGCC ACTTGTGCGC CTTCTCGAAG AACGCCGCCT
5401 GCTGTTCTTG GCTGGCCGAC TTCCACCATT CCGGGCTGGC CGTCATGACG TACTCGACCG
5461 CCAACACAGC GTCCTTGCGC CGCTTCTCTG GCAGCAACTC GCGCAGTCGG CCCATCGCTT
5521 CATCGGTGCT GCTGGCCGCC CAGTGCTCGT TCTCTGGCGT CCTGCTGGCG TCAGCGTTGG
5581 GCGTCTCGCG CTCGCGGTAG GCGTGCTTGA GACTGGCCGC CACGTTGCCC ATTTTCGCCA
5641 GCTTCTTGCA TCGCATGATC GCGTATGCCG CCATGCCTGC CCCTCCCTTT TGGTGTCCAA
5701 CCGGCTCGAC GGGGGCAGCG CAAGGCGGTG CCTCCGGCGG GCCACTCAAT GCTTGAGTAT
5761 ACTCACTAGA CTTTGCTTCG CAAAGTCGTG ACCGCCTACG GCGGCTGCGG CGCCCTACGG
5821 GCTTGCTCTC CGGGCTTCGC CCTGCGCGGT CGCTGCGCTC CCTTGCCAGC CCGTGGATAT
5881 GTGGACGATG GCCGCGAGCG GCCACCGGCT GGCTCGCTTC GCTCGGCCCG TGGACAACCC
5941 TGCTGGACAA GCTGATGGAC AGGCTGCGCC TGCCCACGAG CTTGACCACA GGGATTGCCC
6001 ACCGGCTACC CAGCCTTCGA CCACATACCC ACCGGCTCCA ACTGCGCGGC CTGCGGCCTT
6061 GCCCCATCAA TTTTTTTAAT TTTCTCTGGG GAAAAGCCTC CGGCCTGCGG CCTGCGCGCT
6121 TCGCTTGCCG GTTGGACACC AAGTGGAAGG CGGGTCAAGG CTCGCGCAGC GACCGCGCAG
6181 CGGCTTGGCC TTGACGCGCC TGGAACGACC CAAGCCTATG CGAGTGGGGG CAGTCGAAGG
6241 CGAAGCCCGC CCGCCTGCCC CCCGAGCCTC ACGGCGGCGA GTGCGGGGGT TCCAAGGGGG
6301 CAGCGCCACC TTGGGCAAGG CCGAAGGCCG CGCAGTCGAT CAACAAGCCC CGGAGGGGCC
6361 ACTTTTTGCC GGAGGGGGAG CCGCGCCGAA GGCGTGGGGG AACCCCGCAG GGGTGCCCTT
6421 CTTTGGGCAC CAAAGAACTA GATATAGGGC GAAATGCGAA AGACTTAAAA ATCAACAACT
6481 TAAAAAAGGG GGGTACGCAA CAGCTCATTG CGGCACCCCC CGCAATAGCT CATTGCGTAG
6541 GTTAAAGAAA ATCTGTAATT GACTGCCACT TTTACGCAAC GCATAATTGT TGTCGCGCTG
6601 CCGAAAAGTT GCAGCTGATT GCGCATGGTG CCGCAACCGT GCGGCACCCT ACCGCATGGA
6661 GATAAGCATG GCCACGCAGT CCAGAGAAAT CGGCATTCAA GCCAAGAACA AGCCCGGTCA
6721 CTGGGTGCAA ACGGAACGCA AAGCGCATGA GGCGTGGGCC GGGCTTATTG CGAGGAAACC
6781 CACGGCGGCA ATGCTGCTGC ATCACCTCGT GGCGCAGATG GGCCACCAGA ACGCCGTGGT
6841 GGTCAGCCAG AAGACACTTT CCAAGCTCAT CGGACGTTCT TTGCGGACGG TCCAATACGC
6901 AGTCAAGGAC TTGGTGGCCG AGCGCTGGAT CTCCGTCGTG AAGCTCAACG GCCCCGGCAC
6961 CGTGTCGGCC TACGTGGTCA ATGACCGCGT GGCGTGGGGC CAGCCCCGCG ACCAGTTGCG
7021 CCTGTCGGTG TTCAGTGCCG CCGTGGTGGT TGATCACGAC GACCAGGACG AATCGCTGTT
7081 GGGGCATGGC GACCTGCGCC GCATCCCGAC CCTGTATCCG GGCGAGCAGC AACTACCGAC
7141 CGGCCCCGGC GAGGAGCCGC CCAGCCAGCC CGGCATTCCG GGCATGGAAC CAGACCTGCC
7201 AGCCTTGACC GAAACGGAGG AATGGGAACG GCGCGGGCAG CAGCGCCTGC CGATGCCCGA
7261 TGAGCCGTGT TTTCTGGACG ATGGCGAGCC GTTGGAGCCG CCGACACGGG TCACGCTGCC
7321 GCGCCGGTAG CACTTGGGTT GCGCAGCAAC CCGTAAGTGC GCTGTTCCAG ACTATCGGCT
7381 GTAGCCGCCT CGCCGCCCTA TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA
7441 GCCGGGCCAC CTCGACCTGA ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCGTTTT
7501 TATCAGGCTC TGGGAGGCAG AATAAATGAT CATATCGTCA ATTATTACCT CCACGGGGAG
7561 AGCCTGAGCA AACTGGCCTC AGGCATTTGA GAAGCACACG GTCACACTGC TTCCGGTAGT
7621 CAATAAACCG GTAAACCAGC AATAGACATA AGCGGCTATT TAACGACCCT GCCCTGAACC
7681 GACGACCGGG TcGAATrTGc ETTCGAATTT CTGCCATTCA TCCGCTTATT ATCACTTATT
7741 CAGGCGTAGC ACCAGGCGTT TAAGTCGACC AATAAC
Amino Acid Sequence for modified EPA with signal sequence
Disclosed in WO 2009/104074 (as SEQ ID NO. 6)
Type: PRT
Organism: Artificial
/note=“Description of Artificial Sequence: Synthetic polypeptide”
Length: 643
Sequence:
SEQ ID NO: 25
Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser
1 5 10 15
Ala Ser Ala Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys
20 25 30
Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser
35 40 45
Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gin Gly Val Leu His Tyr
50 55 60
Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp
65 70 75 80
Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly
85 90 95
Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gin Ala
100 105 110
Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys
115 120 125
Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gin
130 135 140
Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu
145 150 155 160
Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu
165 170 175
Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser
180 185 190
Val Val Met Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu
195 200 205
Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val
210 215 220
Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu
225 230 235 240
Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu
245 250 255
Asp Ile Lys Asp Asn Asn Asn Ser Thr Pro Thr Val Ile Ser His Arg
260 265 270
Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln
275 280 285
Ala Cys His Leu Pro Leu Glu Ala Phe Thr Arg His Arg Gln Pro Arg
290 295 300
Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val
305 310 315 320
Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gin Val Asp Gln Val
325 330 335
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu
340 345 350
Ala Ile Arg Glu Gln Pre Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala
355 360 365
Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu
370 375 380
Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala
385 390 395 400
Lys Asp Gln Asn Arg Thr Lys Gly Glu Cys Ala Gly Pro Ala Asp Ser
405 410 415
Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu
420 425 430
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp
435 440 445
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly
450 455 460
Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser
465 470 475 480
Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile
485 490 495
Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr
500 505 510
Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala
515 520 525
Leu Leu Arg Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg
530 535 540
Thr Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg
545 550 555 560
Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro
565 570 575
Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu
580 585 590
Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val
595 600 605
Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile
610 615 620
Ser Ala Leu Pro Asp Tyr Ala Ser Gin Pro Gly Lys Pro Pro Arg Glu
625 630 635 640
Asp Leu Lys
Amino Acid Sequence for PglB
Disclosed in WO 2009/104074 (as SEQ ID NO. 2)
Length: 722
Type: PRT
Organism: Campylobacter jejuni
Sequence:
SEQ ID NO: 26
Met Leu Lys Lys Glu Tyr Leu Lys Asn Pro Tyr Leu Val Leu Phe Ala
1 5 10 15
Met Ile TIe Leu Ala Tyr Val Phe Ser Val Phe Cys Arg Phe Tyr Trp
20 25 30
Val Trp Trp Ala Ser Glu Phe Asn Glu Tyr Phe Phe Asn Asn Gln Leu
35 40 45
Met Ile Ile Ser Asn Asp Gly Tyr Ala Phe Ala Glu Gly Ala Arg Asp
50 55 60
Met Ile Ala Gly Phe His Gln Pro Asn Asp Leu Ser Tyr Tyr Gly Ser
65 70 75 80
Ser Leu Ser Ala Leu Thr Tyr Trp Leu Tyr Lys Ile Thr Pro Phe Ser
85 90 95
Phe Glu Ser Ile Ile Leu Tyr Met Ser Thr Phe Leu Ser Ser Leu Val
100 105 110
Val Ile Pro Thr Ile Leu Leu Ala Asn Glu Tyr Lys Arg Pro Leu Met
115 120 125
Gly Phe Val Ala Ala Leu Leu Ala Ser Ile Ala Asn Ser Tyr Tyr Asn
130 135 140
Arg Thr Met Ser Gly Tyr Tyr Asp Thr Asp Met Leu Val Ile Val Leu
145 150 155 160
Pro Met Phe Ile Leu Phe Phe Met Val Arg Met Ile Leu Lys Lys Asp
165 170 175
Phe Phe Ser Leu Ile Ala Leu Pro Leu Phe Ile Gly Ile Tyr Leu Trp
180 185 190
Trp Tyr Pro Ser Ser Tyr Thr Leu Asn Val Ala Leu Ile Gly Leu Phe
195 200 205
Leu Ile Tyr Thr Leu Ile Phe His Arg Lys Glu Lys Ile Phe Tyr Ile
210 215 220
Ala Val Ile Leu Ser Ser Leu Thr Leu Ser Asn Ile Ala Trp Phe Tyr
225 230 235 240
Gln Ser Ala Ile Ile Val Ile Leu Phe Ala Leu Phe Ala Leu Glu Gln
245 250 255
Lys Arg Leu Asn Phe Met Ile Ile Gly Ile Leu Gly Ser Ala Thr Leu
260 265 270
Ile Phe Leu Ile Leu Ser Gly Gly Val Asp Pro Ile Leu Tyr Gln Leu
275 280 285
Lys Phe Tyr Ile Phe Arg Ser Asp Glu Ser Ala Asn Leu Thr Gln Gly
290 295 300
Phe Met Tyr Phe Asn Val Asn Gln Thr Ile Gln Glu Val Glu Asn Val
305 310 315 320
Asp Leu Ser Glu Phe Met Arg Arg Ile Ser Gly Ser Glu Ile Val Phe
325 330 335
Leu Phe Ser Leu Phe Gly Phe Val Trp Leu Leu Arg Lys His Lys Ser
340 345 350
Met Ile Met Ala Leu Pro Ile Leu Val Leu Gly Phe Leu Ala Leu Lys
355 360 365
Gly Gly Leu Arg Phe Thr Ile Tyr Ser Val Pro Val Met Ala Leu Gly
370 375 380
Phe Gly Phe Leu Leu Ser Glu Phe Lys Ala Ile Met Val Lys Lys Tyr
385 390 395 400
Ser Gln Leu Thr Ser Asn Val Cys Ile Val Phe Ala Thr Ile Leu Thr
405 410 415
Leu Ala Pro Val Phe Ile His Ile Tyr Asn Tyr Lys Ala Pro Thr Val
420 425 430
Phe Ser Gln Asn Glu Ala Ser Leu Leu Asn Gln Leu Lys Asn Ile Ala
435 440 445
Asn Arg Glu Asp Tyr Val Val Thr Trp Ala Ala Tyr Gly Tyr Pro Val
450 455 460
Arg Tyr Tyr Ser Asp Val Lys Thr Leu Val Asp Gly Gly Lys His Leu
465 470 475 480
Gly Lys Asp Asn Phe Phe Pro Ser Phe Ala Leu Ser Lys Asp Glu Gln
485 490 495
Ala Ala Ala Asn Met Ala Arg Leu Ser Val Glu Tyr Thr Glu Lys Ser
500 505 510
Phe Tyr Ala Pro Gln Asn Asp Ile Leu Lys Thr Asp Ile Leu Gln Ala
515 520 525
Met Met Lys Asp Tyr Asn Gln Ser Asn Val Asp Leu Phe Leu Ala Ser
530 535 540
Leu Ser Lys Pro Asp Phe Lys Ile Asp Thr Pro Lys Thr Arg Asp Ile
545 550 555 560
Tyr Leu Tyr Met Pro Ala Arg Met Ser Leu Ile Phe Ser Thr Val Ala
565 570 575
Ser Phe Ser Phe Ile Asn Leu Asp Thr Gly Val Leu Asp Lys Pro Phe
580 585 590
Thr Phe Ser Thr Ala Tyr Pro Leu Asp Val Lys Asn Gly Glu Ile Tyr
595 600 605
Leu Ser Asn Gly Val Val Leu Ser Asp Asp Phe Arg Ser Phe Lys Ile
610 615 620
Gly Asp Asn Val Val Ser Val Asn Ser Ile Val Glu Ile Asn Ser Ile
625 630 635 640
Lys Gln Gly Glu Tyr Lys Ile Thr Pro Ile Asp Asp Lys Ala Gln Phe
645 650 555
Tyr Ile Phe Tyr Leu Lys Asp Ser Ala Ile Pro Tyr Ala Gln Phe Ile
660 665 670
Leu Met Asp Lys Thr Met Phe Asn Ser Ala Tyr Val Gln Met Phe Phe
675 680 685
Leu Gly Asn Tyr Asp Lys Asn Leu Phe Asp Leu Val Ile Asn Ser Arg
690 695 700
Asp Ala Lys Val Phe Lys Leu Lys Ile Tyr Pro Tyr Asp Val Pro Asp
705 710 715 720
Tyr Ala
Nucleotide Sequence for pCC1FOS Empty plasmid
Locus pCC1FOS with MCS cassette
Features Location/Qualifiers
Region 230..256
/label=“pCC1/pEpiFOS fwd”
Region 311..330
/label=“T7 promoter”
Region complement(504..529)
/label=“pCC1pEpiFOS rv”
CDS complement(805..1464)
/label=cat
CDS 1683..2030
/label=redF
CDS 3425..4180
/label=repE
CDS 4759..5934
/label=parA
CDS 5934..6905
/label=parB
ORIGIN
Length: 8171 bp
Type: DNA circular TNA
Organism: Artificial
Sequence:
SEQ ID NO: 27
1 GCGGCCGCAA GGGGTTCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG
61 CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT
121 GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT CAGCTGCGCA ACTGTTGGGA
181 AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC
241 AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC
301 CAGTGAATTG TAATACGACT CACTATAGGG CGAATTCGAG CTCGGTACCC GGGGATCCCA
361 CGTGGCGCGC CACTAGTGCT AGCGACGTCG TGGGATCCTC TAGAGTCGAC CTGCAGGCAT
421 GCAAGCTTGA GTATTCTATA GTCTCACCTA AATAGCTTGG CGTAATCATG GTCATAGCTG
481 TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA
541 AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA
601 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC
661 GAACCCCTTG CGGCCGCCCG GGCCGTCGAC CAATTCTCAT GTTTGACAGC TTATCATCGA
721 ATTTCTGCCA TTCATCCGCT TATTATCACT TATTCAGGCG TAGCAACCAG GCGTTTAAGG
781 GCACCAATAA CTGCCTTAAA AAAATTACGC CCCGCCCTGC CACTCATCGC AGTACTGTTG
841 TAATTCATTA AGCATTCTGC CGACATGGAA GCCATCACAA ACGGCATGAT GAACCTGAAT
901 CGCCAGCGGC ATCAGCACCT TGTCGCCTTG CGTATAATAT TTGCCCATGG TGAAAACGGG
961 GGCGAAGAAG TTOTCCATAT TGGCCACGTT TAAATCAAAA CTGGTGAAAC TCACCCAGGG
1021 ATTGGCTGAG ACGAAAAACA TATTCTCAAT AAACCCTTTA GGGAAATAGG CCAGGTTTTC
1081 ACCGTAACAC GCCACATCTT GCGAATATAT GTGTAGAAAC TGCCGGAAAT CGTCGTGGTA
1141 TTCACTCCAG AGCGATGAAA ACGTTTCAGT TTGCTCATGG AAAACGGTGT AACAAGGGTG
1201 AACACTATCC CATATCACCA GCTCACCGTC TTTCATTGCC ATACGAAATT CCGGATGAGC
1261 ATTCATCAGG CGGGCAAGAA TGTGAATAAA GGCCGGATAA AACTTGTGCT TATTTTTCTT
1321 TACGGTCTTT AAAAAGGCCG TAATATCCAG CTGAACGGTC TGGTTATAGG TACATTGAGC
1381 AACTGACTGA AATGCCTCAA AATGTTCTTT ACGATGCCAT TGGGATATAT CAACGGTGGT
1441 ATATCCAGTG ATTTTTTTCT CCATTTTAGC TTCCTTAGCT CCTGAAAATC TCGATAACTC
1501 AAAAAATACG CCCGGTAGTG ATCTTATTTC ATTATGGTGA AAGTTGGAAC CTCTTACGTG
1561 CCGATCAACG TCTCATTTTC GCCAAAAGTT GGCCCAGGGC TTCCCGGTAT CAACAGGGAC
1621 ACCAGGATTT ATTTATTCTG CGAAGTGATC TTCCGTCACA GGTATTTATT CGCGATAAGC
1681 TCATGGAGCG GCGTAACCGT CGCACAGGAA GGACAGAGAA AGCGCGGATC TGGGAAGTGA
1741 CGGACAGAAC GGTCAGGACC TGGATTGGGG AGGCGGTTGC CGCCGCTGCT GCTGACGGTG
1801 TGACGTTCTC TGTTCCGGTC ACACCACATA CGTTCCGCCA TTCCTATGCG ATGCACATGC
1861 TGTATGCCGG TATACCGCTG AAAGTTCTGC AAAGCCTGAT GGGACATAAG TCCATCAGTT
1921 CAACGGAAGT CTACACGAAG GTTTTTGCGC TGGATGTGGC TGCCCGGCAC CGGGTGCAGT
1981 TTGCGATGCC GGAGTCTGAT GCGGTTGCGA TGCTGAAACA ATTATCCTGA GAATAAATGC
2041 CTTGGCCTTT ATATGGAAAT GTGGAACTGA GTGGATATGC TGTTTTTGTC TGTTAAACAG
2101 AGAAGCTGGC TGTTATCCAC TGAGAAGCGA ACGAAACAGT CGGGAAAATC TCCCATTATC
2161 GTAGAGATCC GCATTATTAA TCTCAGGAGC CTGTGTAGCG TTTATAGGAA GTAGTGTTCT
2221 GTCATGATGC CTGCAAGCGG TAACGAAAAC GATTTGAATA TGCCTTCAGG AACAATAGAA
2281 ATCTTCGTGC CGTGTTACGT TGAAGTGGAG CGGATTATGT CAGCAATGGA CAGAACAACC
2341 TAATGAACAC AGAACCATGA TGTGGTCTGT CCTTTTACAG CCAGTAGTGC TCGCCGCAGT
2401 CGAGCGACAG GGCGAAGCCC TCGGCTGGTT GCCCTCGCCG CTGGGCTGGC GGCCGTCTAT
2461 GGCCCTGCAA ACGCGCCAGA AACGCCGTCG AAGCCGTGTG CGAGACACCG CGGCCGGCCG
2521 CCGGCGTTGT GGATACCTCG CGGAAAACTT GGCCCTCACT GACAGATGAG GGGCGGACGT
2581 TGACACTTGA GGGGCCGACT CACCCGGCGC GGCGTTGACA GATGAGGGGC AGGCTCGATT
2641 TCGGCCGGCG ACGTGGAGCT GGCCAGCCTC GCAAATCGGC GAAAACGCCT GATTTTACGC
2701 GAGTTTCCCA CAGATGATGT GGACAAGCCT GGGGATAAGT GCCCTGCGGT ATTGACACTT
2761 GAGGGGCGCG ACTACTGACA GATGAGGGGC GCGATCCTTG ACACTTGAGG GGCAGAGTGC
2821 TGACAGATGA GGGGCGCACC TATTGACATT TGAGGGGCTG TCCACAGGCA GAAAATCCAG
2881 CATTTGCAAG GGTTTCCGCC CGTTTTTCGG CCACCGCTAA CCTGTCTTTT AACCTGCTTT
2941 TAAACCAATA TTTATAAACC TTGTTTTTAA CCAGGGCTGC GCCCTGTGCG CGTGACCGCG
3001 CACGCCGAAG GGGGGTGCCC CCCCTTCTCG AACCCTCCCG GTCGAGTGAG CGAGGAAGCA
3061 CCAGGGAACA GCACTTATAT ATTCTGCTTA CACACGATGC CTGAAAAAAC TTCCCTTGGG
3121 GTTATCCACT TATCCACGGG GATATTTTTA TAATTATTTT TTTTATAGTT TTTAGATCTT
3181 CTTTTTTAGA GCGCCTTGTA GGCCTTTATC CATGCTGGTT CTAGAGAAGG TGTTGTGACA
3241 AATTGCCCTT TCAGTGTGAC AAATCACCCT CAAATGACAG TCCTGTCTGT GACAAATTGC
3301 CCTTAACCCT GTGACAAATT GCCCTCAGAA GAAGCTGTTT TTTCACAAAG TTATCCCTGC
3361 TTATTGACTC TTTTTTATTT AGTGTGACAA TCTAAAAACT TGTCACACTT CACATGGATC
3421 TGTCATGGCG GAAACAGCGG TTATCAATCA CAAGAAACGT AAAAATAGCC CGCGAATCGT
3481 CCAGTCAAAC GACCTCACTG AGGCGGCATA TAGTCTCTCC CGGGATCAAA AACGTATGCT
3541 GTATCTGTTC GTTGACCAGA TCAGAAAATC TGATGGCACC CTACAGGAAC ATGACGGTAT
3601 CTGCGAGATC CATGTTGCTA AATATGCTGA AATATTCGGA TTGACCTCTG COGAAGCCAG
3661 TAAGGATATA CGGCAGGCAT TGAAGAGTTT CGCGGGGAAG GAAGTGGTTT TTTATCGCCC
3721 TGAACAGGAT GCCGGCGATG AAAAAGGCTA TGAATCTTTT CCTTGGTTTA TCAAACGTGC
3781 GCACAGTCCA TCCAGAGGGC TTTACAGTGT ACATATCAAC CCATATCTCA TTCCCTTCTT
3841 TATCGGGTTA CAGAACCGGT TTACGCAGTT CGGCTTAGTG GAAACAAAAG AAATCACCAA
3901 TCCGTATCCC ATGCGTTTAT ACGAATCCCT GTGTCAGTAT CGTAAGCCGG ATGGCTCAGG
3961 CATCGTCTCT CTGAAAATCG ACTGGATCAT AGAGCGTTAC CAGCTGCCTC AAAGTTACCA
4021 GCGTATGCCT GACTTCCGCC GCCGCTTCCT GCAGGTCTGT GTTAATGAGA TCAACAGCAG
4081 AACTCCAATG CGCCTCTCAT ACATTGAGAA AAAGAAAGGC CGCCAGACGA CTCATATCGT
4141 ATTTTCCTTC CGCGATATCA CTTCCATGAC GACAGGATAG TCTGAGGGTT ATCTGTCACA
4201 GATTTGAGGG TGGTTCGTCA CATTTGTTCT GACCTACTGA GGGTAATTTG TCACAGTTTT
4261 GCTGTTTCCT TCAGCCTGCA TGGATTTTCT CATACTTTTT GAACTGTAAT TTTTAAGGAA
4321 GCCAAATTTG AGGGCAGTTT GTCACAGTTG ATTTCCTTCT CTTTCCCTTC GTCATGTGAC
4381 CTGATATCGG GGGTTAGTTC GTCATCATTG ATGAGGGTTG ATTATCACAG TTTATTACTC
4441 TGAATTGGCT ATCCGCGTGT GTACCTCTAC CTGGAGTTTT TCCCACGGTG GATATTTCTT
4501 CTTGCGCTGA GCGTAAGAGC TATCTGACAG AACAGTTCTT CTTTGCTTCC TCGCCAGTTC
4561 GCTCGCTATG CTCGGTTACA CGGCTGCGGC GAGCGCTAGT GATAATAAGT GACTGAGGTA
4621 TGTGCTCTTC TTATCTCCTT TTGTAGTGTT GCTCTTATTT TAAACAACTT TGCGGTTTTT
4681 TGATGACTTT GCGATTTTGT TGTTGCTTTG CAGTAAATTG CAAGATTTAA TAAAAAAACG
4741 CAAAGCAATG ATTAAAGGAT GTTCAGAATG AAACTCATGG AAACACTTAA CCAGTGCATA
4801 AACGCTGGTC ATGAAATGAC GAAGGCTATC GCCATTGCAC AGTTTAATGA TGACAGCCCG
4861 GAAGCGAGGA AAATAACCCG GCGCTGGAGA ATAGGTGAAG CAGCGGATTT AGTTGGGGTT
4921 TCTTCTCAGG CTATCAGAGA TGCCGAGAAA GCAGGGCGAC TACCGCACCC GGATATGGAA
4981 ATTCGAGGAC GGGTTGAGCA ACGTGTTGGT TATACAATTG AACAAATTAA TCATATGCGT
5041 GATGTGTTTG GTACGCGATT GCGACGTGCT GAAGACGTAT TTCCACCGGT GATCGGGGTT
5101 GCTGCCCATA AAGGTGGCGT TTACAAAACC TCAGTTTCTG TTCATCTTGC TCAGGATCTG
5161 GCTCTGAAGG GGCTACGTGT TTTGCTCGTG GAAGGTAACG ACCCCCAGGG AACAGCCTCA
5221 ATGTATCACG GATGGGTACC AGATCTTCAT ATTCATGCAG AAGACACTCT CCTGCCTTTC
5281 TATCTTGGGG AAAAGGACGA TGTCACTTAT GCAATAAAGC CCACTTGCTG GCCGGGGCTT
5341 GACATTATTC CTTCCTGTCT GGCTCTGCAC CGTATTGAAA CTGAGTTAAT GGGCAAATTT
5401 GATGAAGGTA AACTGCCCAC CGATCCACAC CTGATGCTCC GACTGGCCAT TGAAACTCTT
5461 GCTCATGACT ATGATGTCAT AGTTATTGAC AGCGCGCCTA ACCTGGGTAT CGGCACGATT
5521 AATGTCGTAT GTGCTGCTGA TGTGCTGATT GTTCCCACGC CTGCTGAGTT GTTTGACTAC
5581 ACCTCCGCAC TGCAGTTTTT CGATATGCTT CGTGATCTGC TCAAGAACGT TGATCTTAAA
5641 GGGTTCGAGC CTGATGTACG TATTTTGCTT ACCAAATACA GCAATAGTAA TGGCTCTCAG
5701 TCCCCGTGGA TGGAGGAGCA AATTCGGGAT GCCTGGGGAA GCATGGTTCT AAAAAATGTT
5761 GTACGTGAAA CGGATGAAGT TGGTAAAGGT CAGATCCGGA TGAGAACTGT TTTTGAACAG
5821 GCCATTGATC AACGCTCTTC AACTGGTGCC TGGAGAAATG CTCTTTCTAT TTGGGAACCT
5881 GTCTGCAATG AAATTTTCGA TCGTCTGATT AAACCACGCT GGGAGATTAG ATAATGAAGC
5941 GTGCGCCTGT TATTCCAAAA CATACGCTCA ATACTCAACC GGTTGAAGAT ACTTCGTTAT
6001 CGACACCAGC TGCCCCGATG GTGGATTCGT TAATTGCGCG CGTAGGAGTA ATGGCTCGCG
6061 GTAATGCCAT TACTTTGCCT GTATGTGGTC GGGATGTGAA GTTTACTCTT GAAGTGCTCC
6121 GGGGTGATAG TGTTGAGAAG ACCTCTCGGG TATGGTCAGG TAATGAACGT GACCAGGAGC
6181 TGCTTACTGA GGACGCACTG GATGATCTCA TCCCTTCTTT TCTACTGACT GGTCAACAGA
6241 CACCGGCGTT CGGTCGAAGA GTATCTGGTG TCATAGAAAT TGCCGATGGG AGTCGCCGTC
6301 GTAAAGCTGC TGCACTTACC GAAAGTGATT ATCGTGTTCT GGTTGGCGAG CTGGATGATG
6361 AGCAGATGGC TGCATTATCC AGATTGGGTA ACGATTATCG CCCAACAAGT GCTTATGAAC
6421 GTGGTCAGCG TTATGCAAGC CGATTGCAGA ATGAATTTGC TGGAAATATT TCTGCGCTGG
6481 CTGATGCGGA AAATATTTCA CGTAAGATTA TTACCCGCTG TATCAACACC GCCAAATTGC
6541 CTAAATCAGT TGTTGCTCTT TTTTCTCACC CCGGTGAACT ATCTGCCCGG TCAGGTGATG
6601 CACTTCAAAA AGCCTTTACA GATAAAGAGG AATTACTTAA GCAGCAGGCA TCTAACCTTC
6661 ATGAGCAGAA AAAAGCTGGG GTGATATTTG AAGCTGAAGA AGTTATCACT CTTTTAACTT
6721 CTGTGCTTAA AACGTCATCT GCATCAAGAA CTAGTTTAAG CTCACGACAT CACTTTGCTC
6781 CTGGAGCGAC AGTATTGTAT AAGGGCCATA AAATGGTGCT TAACCTGGAC AGGTCTCGTG
6841 TTCCAACTGA GTGTATAGAG AAAATTGAGG CCATTCTTAA GGAACTTGAA AAGCCAGCAC
6901 CCTGATGCGA CCACGTTTTA GTTTACTTTT ATCTGTCTTT ACTTAATGTC CTTTGTTACA
6961 GGCCAGAAAG CATAACTGGC CTGAATATTC TCTCTGGGCC CACTGTTCCA CTTGTATCGT
7021 CGGTCTGATA ATCAGACTGG GACCACGGTC CCACTCGTAT CGTCGGTCTG ATTATTAGTC
7081 TGGGACCACG GTCCCACTCG TATCGTCGGT CTGATTATTA GTCTGGGACC ACGGTCCCAC
7141 TCGTATCGTC GGTCTGATAA TCAGACTGGG ACCACGGTCC CACTCGTATC GTCGGTCTGA
7201 TTATTAGTCT GGGACCATGG TCCCACTCGT ATCGTCGGTC TGATTATTAG TCTGGGACCA
7261 CGGTCCCACT CGTATCGTCG GTCTGATTAT TAGTCTGGAA CCACGGTCCC ACTCGTATCG
7321 TCGGTCTGAT TATTAGTCTG GGACCACGGT CCCACTCGTA TCGTCGGTCT GATTATTAGT
7381 CTGGGACCAC GATCCCACTC GTGTTGTCGG TCTGATTATC GGTCTGGGAC CACGGTCCCA
7441 CTTGTATTGT CGATCAGACT ATCAGCGTGA GACTACGATT CCATCAATGC CTGTCAAGGG
7501 CAAGTATTGA CATGTCGTCG TAACCTGTAG AACGGAGTAA CCTCGGTGTG CGGTTGTATG
7561 CCTGCTGTGG ATTGCTGCTG TGTCCTGCTT ATCCACAACA TTTTGCGCAC GGTTATGTGG
7621 ACAAAATACC TGGTTACCCA GGCCGTGCCG CCACGTTAAC CGGGCTGCAT CCGATGCAAG
7681 TGTGTCGCTG TCGACGAGCT CGCGAGCTCG GACATGAGGT TGCCCCGTAT TCAGTGTCGC
7741 TGATTTGTAT TGTCTGAAGT TGTTTTTACG TTAAGTTGAT GCAGATCAAT TAATACGATA
7801 CCTGCGTCAT AATTGATTAT TTGACGTGGT TTGATGGCCT CCACGCACGT TGTGATATGT
7861 AGATGATAAT CATTATCACT TTACGGGTCC TTTCCGGTGA TCCGACAGGT TACGGGGCGG
7921 CGACCTCGCG GGTTTTCGCT ATTTATGAAA ATTTTCCGGT TTAAGGCGTT TCCGTTCTTC
7981 TTCGTCATAA CTTAATGTTT TTATTTAAAA TACCCTCTGA AAAGAAAGGA AACGACAGGT
8041 GCTGAAAGCG AGCTTTTTGG CCTCTGTCGT TTCCTTTCTC TGTTTTTGTC CGTGGAATGA
8101 ACAATGGAAG TCCGAGCTCA TCGCTAATAA CTTCGTATAG CATACATTAT ACGAAGTTAT
8161 ATTCGATCCA C
Nucleotide Sequence for pCC1FOS cut (pFOS) and
S. flexneri 6 O-antigen without Z3206
Locus pFOS cut and O-antige cut (-Z3206)
Definition Ligation of inverted pCC1FOS with MCS cassette cut with
NheI and into S. flexneri 6 O antigen cluster amplified with
galFNheI and wzzAscI cut with NheI and AscI
FEATURES Location/Qualifiers
CDS 3..411
/label=′galF
CDS 784..1869
/label=rmlB
CDS 1869..2768
/label=rmlD
CDS 2826..3704
/label=rmlA
CDS 3709..4266
/label=rmlC
CDS 4263..5495
/label=wzx
CDS 5551..6738
/label=wzy
CDS 6755..7624
/label=wfbY
CDS 7621..8454
/label=wfbZ
CDS 8559..9965
/label=gnd
CDS 10187..11380
/label=ugd
CDS complement(11416..12450)
/label=uge
CDS 12802..12828
/label=wzz′
Region complement(12868..12887)
/label=“T7 promoter”
Region complement(12942..12968)
/label=“pCC1/pEpiRDS fwd”
CDS complement(14460..15431)
/label=parB
CDS complement(15431..16606)
/label=parA
CDS complement(7185..17940)
/label=repE
CDS complement(19335..19682)
/label=redF
CDS 19901..20560
/label=cat
Region 20836..20861
/label=“pCC1pEpiFOS rv”
Length: 20982 bp
Type: DNA circular UNA
Sequence:
SEQ ID NO: 28
1 CTAGCGGCAA AACGTATGCC GGGTGACCTC TCTGAATACT CCGTCATCCA GACCAAAGAA
61 CCGCTGGATC GCGAAGGTAA AGTCAGCCGC ATTGTTGAAT TTATCGAAAA ACCGGATCAG
121 CCGCAGACGC TGGACTCAGA CATCATGGCC GTTGGTCGCT ATGTGCTTTC TGCCGATATT
181 TGGCCGGAAC TTGAACGTAC TCAGCCTGGT GCATGGGGAC GTATTCAGCT GACTGATGCC
241 ATTGCCGAGC TGGCGAAAAA ACAGTCCGTT GATGCAATGC TGATGACCGG CGACAGCTAC
301 GACTGCGGTA AAAAAATGGG CTATATGCAG GCGTTTGTGA AGTATGGGCT GCGCAACCTG
361 AAAGAAGGGG CGAAGTTCCG TAAAGGTATT GAGAAGCTGT TAAGCGAATA ATGAAAATCT
421 GACCGGATGT AACGGTTGAT AAGAAAATTA TAACGGCAGT GAAGATTCGT GGTGAAAGTA
481 ATTTGTTGCG AATATTCCTG CCGTTGTTTT ATATAAACAA TCAGAATAAC AACGAGTTAG
541 CAATAGGATT TTAGTCAAAG TTTTCCAGGA TTTTCCTTGT TTCCAGAGCG GATTGGTAAG
601 ACAATTAGCT TTTGAATTTT TCGGGTTTAG CGCGAGTGGG TAACGCTCGT CACATCGTAG
661 GCATGCATGC AGTGCTCTGG TAGCTGTAAA GCCAGGGGCG GTAGCGTGCA TTAATACTTC
721 TATTAATCAA ACTGAGAGCC GCTTATTTCA CAGCATGCTC TGAAGCAATA TGGAATAAAT
781 TAGGTGAAAA TACTTGTTAC TGGTGGCGCA GGATTTATTG GTTTTGCTGT AGTTCGTCAC
841 ATTATAAATA ATACGCAGGA TAGTGTTGTT AATGTCGATA AATTAACGTA CGCCGGAAAC
901 CTGGAATCAC TTGCTGATGT TTCTGATTCT GAACGCTATG TTTTTGAACA TGCGGATATT
961 TGCGATGCAG CTGCAATGGC ACGGATTTTT GCTCAGCATC AGCCAGATGC AGTGATGCAC
1021 CTGGCTGCTG AAAGCCATGT TGACCGTTCA ATTACAGGTC CTGCGGCATT TATTGAAACC
1081 AATATTGTTG GTACATATGT CCTTTTGGAA GCCGCTCGCA ATTATTGGTC TGCTCTTGAT
1141 AGCGACAAGA AAACTAGATT CCGTTTTCAT CATATTTCTA CTGACGAAGT CTATGGTGAT
1201 TTGCCTCATC CTGACGAGGT AAATAATACA GAAGAATTAC CCTTATTTAC AGAGACAACA
1261 GCTTACGCGC CAAGCAGCCC TTATTCCGCT TCAAAAGCAT CCAGCGATCA TTTAGTCCGC
1321 GCGTGGAAAC GTACCTATGG TTTACCAACC ATTGTGACTA ATTGCTCTAA TAATTATGGT
1381 CCTTATCATT TCCCGGAAAA ATTGATTCCA TTGGTTATTC TGAATGCTCT GGAAGGTAAG
1441 GCATTACCTA TTTATGGCAA AGGGGATCAA ATTCGTGACT GGCTGTATGT TGAAGATCAT
1501 GCGCGTGCGT TATATACCGT CGTAACCGAA GGTAAAGCGG GTGAAACTTA TAACATTGGT
1561 GGACACAACG AAAAGAAAAA CATCGATGTA GTGCTCACTA TTTGTGATTT GCTGGATGAG
1621 ATTGTACCGA AAGAGAAATC TTACCGCGAG CAAATTACTT ATGTTGCCGA TCGCCCGGGA
1681 CACGATCGCC GTTATGCGAT TGATGCAGAG AAGATTAGCC GCGAATTGGG CTGGAAACCG
1741 CAGGAAACGT TTGAGAGCGG GATTCGGAAG ACATTGGAAT GGTACCTGTC CAATACAAAA
1801 TGGGTTGATA ATGTGAAAAG TGGTGCTTAT CAATCGTGGA TTGAACAGAA CTATGAGGGC
1861 CGCCAGTAAT GAATATCCTC CTTTTCGGCA AAACAGGGCA GGTAGGTTGG GAACTACAGC
1921 GTGCTCTGGC ACCTTTGGGT AATTTGATTG CTCTTGATGT TCACTCCACT GATTATTGTG
1981 GTGATTTTAG TAATCCTGAA GGTGTAGCTG AAACAGTCAA AAGAATTCGA CCTGATGTTA
2041 TTGTTAATGC TGCGGCTCAC ACCGCAGTAG ATAAGGCTGA GTCAGAACCC GAATTTGCAC
2101 AATTACTCAA TGCGACTAGT GTTGAATCAA TTGCAAAAGA GGCTAATGAA GTTGGGGCTT
2161 GGGTAATTCA TTACTCAACT GACTACGTAT TCCCTGGAAA TGGCGACACG CCATGGCTGG
2221 AGACGGATGC AACCGCACCG CTAAATGTTT ACGGTGAAAC CAAGTTAGCC GGAGAAAAAG
2281 CGTTACAGGA ACATTGCGCG AAGCATCTTA TTTTCCGTAC CAGCTGGGTA TACGCAGCTA
2341 AAGGAAATAA CTTCGCCAAA ACGATGTTGC GTCTGGCAAA AGAGCGCGAA GAACTGGCTG
2401 TGATAAATGA TCAATTTGGT GCGCCAACAG GTGCTGAGCT GCTGGCTGAT TGTACGGCAC
2461 ATGCTATTCG TGTGGCACTG AATAAACCGG AAGTCGCAGG TTTGTACCAT CTGGTAGCCA
2521 GTGGTACCAC AACCTGGCAC GATTATGCTG CGCTGGTTTT TGAAGAGGCG CGCAAAGCAG
2581 GTATTCCCCT TGCACTCAAC AAGCTCAACG CAGTACCAAC AACAGCCTAT CCTACACCAG
2641 CTCGTCGTCC ACATAACTCT CGCCTTAATA CAGAAAAATT TCAGCAGAAC TTTGCGCTTG
2701 TCTTGCCTGA CTGGCAGGTT GGTGTGAAAC GAATGCTCAA CGAATTAATT ACGACTACAG
2761 CAATTTAATA GTTTTTGCAT CTTGTTCGTG ATGGTGGAGC AAGATGAATT AAAAGGAATG
2821 ATGAAATGAA AACGCGTAAA GGTATTATTT TAGCGGGTGG TTCTGGTACA CGTCTTTATC
2881 CTGTGACTAT GGCTGTCAGT AAACAGCTAT TACCTATTTA TGATAAGCCG ATGATCTATT
2941 ACCCGCTCTC TACACTGATG TTGGCGGGTA TTCGCGATAT TCTGATTATT AGTACGCCAC
3001 AGGATACTCC TCGTTTTCAA CAACTGCTAG GTGACGGTAG CCAGTGGGGG CTAAATCTTC
3061 AGTACAAAGT GCAACCGACT CCAGATGGGC TTGCGCAGGC GTTTATTATC GGTGAAGAGT
3121 TTATCGGTGG TGATGATTGT GCTTTGGTTC TTGGTGATAA TATCTTCTAC GGTCATGATC
3181 TGCCGAAGTT AATGGATGTC GCTGTTAACA AAGAAAGTGG TGCAACGGTA TTTGCCTATC
3241 ACGTTAATGA TCCTGAACGC TACGGCGTCG TTGAGTTTGA TAAAAACGGT ACGGCAATAA
3301 GCCTGGAAGA AAAACCGCTA CAACCAAAAA GTAATTATGC GGTAACCGGG CTTTATTTCT
3361 ATGATAACGA CGTTGTCGAA ATGGCGAAAA ACCTTAAGCC TTCTGCCCGT GGTGAACTGG
3421 AAATTACCGA TATTAACCGT ATTTATATGG AACAGGGGCG TTTATCCGTT GCCATGATGG
3481 GGCGTGGTTA TGCATGGCTG GATACGGGGA CACATCAGAG TCTTATTGAA GCAAGCAACT
3541 TCATTGCCAC CATTGAAGAG CGCCAGGGAC TAAAGGTTTC CTGCCCAGAA GAAATTGCTT
3601 ACCGTAAAGG GTTTATTGAT GCTGAACAGG TGAAAGCATT AGCGGAGCCG CTGAAAAAAA
3661 ATGCTTATGG ACAGTATCTG CTGAAAATGA TTAAAGGTTA TTAATAAAAT GAACGTAATT
3721 AAAACAGAAA TTCCTGATGT GTTAATTTTC GAGCCGAAAG TTTTTGGTGA TGAGCGTGGT
3781 TTCTTTATGG AAAGCTTTAA TCAGAAAGTT TTCGAAGAAG CTGTAGGACG TAAGGTTGAA
3841 TTTGTTCAGG ATAACCATTC GAAGTCTAGT AAAGGTGTTT TACGCGGGCT GCATTATCAG
3901 TTAGAACCTT ATGCGCAAGG GAAACTGGTA CGTTGCGTTG TTGGTGAGGT TTTTGATGTA
3961 GCTGTTGATA TTCGTAAATC GTCGCCTACC TTTGGTAAAT GGGTTGGGGT GAATTTATCT
4021 GCTGAGAATA AGCGGCAATT GTGGATCCCT GAGGGATTTG CACATGGTTT TTTGGTGCTG
4081 AGCGAGACTG CGGAATTTTT ATATAAAACG ACGAACTATT ATCATCCTGA TAGTGATAGA
4141 GGGATTGTAT GGAATGATCC TATTCTGAGC ATAAAATGGC CGACGATAGA ACATAATAAT
4201 TATATTTTAT CGATTAAAGA TGCAAGGGCT AAAGAATTGC ATAACATGAA GGAATTATTT
4261 TTGTGAGTAT TGTAAAGAAT ACTTTATGGA ATATAAGTGG GTATATTATA CCATCATTAA
4321 TAGCAATTCC TGCGTTAGGT ATACTGTCTA GAATTCTAGG GACCGAGCAA TTTGGCCTTT
4381 TTACGTTAGC TATTGCCTTA GTTGGATATG CAAGTATTTT TGATGCTGGA TTGACCAGAG
4441 CTGTTATAAG AGAAGTATCA ATATATAAAA ATGTTCATAA AGAATTAAGA GCGATCATTT
4501 CAACTTCAAC GGTAATTCTA ACTATATTGG GCTTGATTGG CGGTAGTGTA CTATTTTTGA
4561 GTAGCAATGT AATTGTTAAA TTATTAAACA TTAACGCGAA TCATGTTGTA GAATCTGTCA
4621 AAGCAATATA TATTATTTCA GCTACCATAC CCTTATACTT GTTAAACCAA GTCTGGTTGG
4681 GGATTTTTGA GGGGATGGAA AAGTTCAGAA AAGTAAATTT AATAAAATCA ATTAACAACT
4741 CTTTTGTGGC TGGATTACCA GTGATTTTCT GTTTTTTTCA TGGAGGATTA CTAAGTGCTA
4801 TATATGGTTT AGTTATGGCA AGAGTCTTAT CACTTATAGT GACCTTTATA TTTAGTCGAA
4861 AACTAATAAT ATCATCTGGG CTGTCTGTAA AAATTGTAAC AGTTAAAAGA TTAATCGGCT
4921 TTGGAAGCTG GATAACAGTT AGCAATATTA TTAGCCCTAT TATGACATAT ATGGATCGTT
4981 TTATTCTTTC ACACATTGTG GGGGCTGATA AAGTTTCTTT TTATACTGCT CCGTCTGAAG
5041 GTATACAACG CTTAACGATA TTACCAAGTG CGTTGTCCAG AGCTATTTTT CCAAGATTAA
5101 GTTCAGAATT GCAATCGGTA AAGCAAACTA AAATATTATC ATATTTTATA ATGGTTATTG
5161 GTATACTTCC AATTGTAATG TTGATAATTA TTTTATCAGA TTTTATAATG TCCGCTTGGA
5221 TGGGACCTAC ATATCATGGG ACGCCAGGTA TAGTATTAAA AATTCTTGCA ATAGGTTTCT
5281 TTTTTAATTG CATTGCACAA ATCCCATTTG TTTCAGTTCA GGCTAGTGGA AGATCAAAAA
5341 TTACAGCTAT TATTCATTTG CTCGAAGTTA TCCCATATTT ATGCATATTA TATATTTTTA
5401 TTTATCATTG GGGAATTGTT GGAGCCGCAA TAGCATGGTC TGTAAGAACA TCGTTAGATT
5461 TTTTGATATT ATTATTAATT GATACGAAAT ATTAATAGCG AATTGATTTT AGGGATTACT
5521 TCCTCAAGCC CATCTAATTA GAGTGCAAAC ATGACTTCTG ATTTTTATAA CTCAAAAGAC
5581 AAAAGTTTAA GTGTTCTTTT GTTTTTTGGG TTTATATTTT TCCTTACACG TAGCTTTCCA
5641 TTTATTCAAT ATAGTTOGAT TATGGAGGGG TTTTTATGTC TTTGTATCAT GTCATTTACA
5701 AAGAAAATTG CAAACGGAAT ATATCACTAT CCTGTTATTT TAATATTTCT ATTAGCTCTT
5761 TTTATAAATT TTATTTATTC CTATATCAAG GGTAACGATA TAGCGATAAT AATTAGGTTT
5821 TATATTATCA TATTATTTAT ATTATGTGCT TATTTCTGCT CTTATGGAAC CATCTCGATT
5881 GTTAAAATAT TTTTATATTT AATGGTATTA CAGGCGGTTA TTATATCCAT CATTAGTATT
5941 TATATGACAA AAACATATGG TATTGGTGAT TATTCAGCAC TAAGACATTA TTTTTTGGAG
6001 AATGATTATG GTGATGTTTA TACATATGGA AGTGGTTTCT ATAGAGTTCA AATTAAAGGA
6061 AATGCTCTCA TTCCATTTGC CTTTATGTTG CATATAGTCA TAAAAGATTA TTTCTATTAT
6121 CGATTCAAAA ATACAATAAC CGTTATTCTG GCTATAGGTA CTATAGTGGC TGGTAATTTT
6181 GCATATTTTG TTTCGATATG CTTGTTTTTT ATGTATATTA TACTATGTTC TAAATCTAAC
6241 TCACGATACG CTAAATTAAG GAAAATTATT TTTGGGGTTT TTCTTACTGT GATTCTCCCT
6301 TTTTTTATTA CATATTCAAT TGAGTTGATA ATCATGAAAT CAAATGGAGC TGATTCTTCT
6361 TTAGGAGTTA GATGGGATCA GTTTACTGTA TTAATTAATG ATCTTACAGA GTCTGTATCA
6421 AATTTTGTTA TAGGTTCTGG TTTGGGTAAT GTCATCAAAA TTCAAACTCC TATCCGTGAT
6481 TATAGTGCAT ATATATATTA TGAATTGCAG TCAGTTTATT TTTTAAATCA ACTTGGCGTT
6541 ATTTTATTTA CTTTGTTTTT ATTAATTAAT CTCCTTCTCA CGATTAAAAT CATAAAATAC
6601 AGTGAGTTGT GTGTGCTATA TTTTCTATAT GTTTCTTATG CAATTACTAA TCCTTATATT
6661 TTAGACTCTA ACCATGTTGC TGTAATAATT GTATTAGTGA CATTAAGTAA TGTTCTAAAA
6721 AAGATGAAAG CTAAATGAAG GTTTTAAGGT GAAGATGGAC ACTGTATATG CCGTTTTGGT
6781 TGCTTACAAC CCAGAACATA ATGATTTAAA AAATGCGGTT GAATTATTGT TGAGACAAGT
6841 TACTAAAGTT GTCGTTTGCA ATAACTCTAC AAATGGTTAT AAATATGCTG AAAATTCTTC
6901 AGGCGATGTA AAAATATTCA ATTTCAATGA TAATTTAGGC ATAGCAGAAG CCCAAAGTAT
6961 AGGAATGAAA TGGGCTTTTG AAAATGGCGC TGATTTTATA TTGCAAATGG ATCAGGATAG
7021 TATTCCTGAT CCTAAGATGG TAGAGCAGTT ACTTACTTGT TACAAAAAAT TGCTTAAACA
7081 AAATGTCAAT GTTGGTTTAG TTGGTTCACA AGATTTTGAT AAAGTAACTG GTGAATTAAA
7141 TAAAGCAAGG GTAAAAAAAG GGAAACCACT TACAGAAGTT TATTATGAGG TAGATAGTAC
7201 AlTAAGTTCT GGCAGTCTAA TACCAAAAAA TAGTTGGTTG ATTGTTGGAG GAATGAAAGA
7261 TGAGCTTTTT ATCGATGCGG TAGACCATGA ATATTGTTGG AGATTAAGAG CTGCTGGGTT
7321 TAAAGTAATT AGGAATAAAA ATGCGTTACT TGCACATAGA CTTGGAGATG GGCGATTTAA
7381 GATCTTAAAT ATTCTTTCTG TCGGTTTGCC AAGCCCATTT CGTCATTATT ATGCTACTCG
7441 AAATATCTTT CTTTTATTAA ATAAAAATTA TGTACCCATC TACTGGAAAA TTTCTAGTCT
7501 GGTTAAATTA ATTGGAAAGG TTTTTTTATA TCCTATTTTC CTTCCAAATG GTAATAAAAG
7561 GTTATATTTT TTTTTAAAAG GCATTAATGA CGGTTTAATG GGTCGAAGTG GTAAAATGAA
7621 ATGAATCATA GATTAGAAAA ATTCTCAGTT TTAATTAGCA TTTATAAAAA TGATCTACCG
7681 CAATTTTTTG AGGTGGCTCT ACGCTCTATT TTTCACGATC AAACACTTAA GCCAGATCAA
7741 ATAGTAATTG TTGCAGATGG AGAACTCCAT CAAACACACA TCGATATTAT AAATTCATTC
7801 ATTGATGATG TTGGCAATAA AATAGTAACA TTTGTACCTT TACCTAGAAA TGTTGGATTG
7861 GCTAATGCCT TAAATGAAGG ATTAAAGGCT TGTAGGAATG AGTTAGTGGC AAGAATGGAT
7921 GCTGATGATA TTTCTTTGCC TCATCGGTTT GAGAAACAAA TTTCTTTTAT GATTAATAAT
7981 TCAGAAATAG ATGTATGTGG CAGTTTTATT GATGAAATTG AAACTGTTAC TGAGGAGTTT
8041 ATTTCAACAC GCAAAGTGCC TCTCGAACAT AGAGAAATAG TTAAATTCGC GAGGAAACGA
8101 AGCGCAGTTA GCCATCCTTC TGTAATTTTT AGAAAGAATA CAGTATTAGC TGTTGGTGGT
8161 TATCCTCCAT TCAGAAAATC TCAAGATTTT GCATTGTGGA GCCTATTAAT TGTACATAAT
8221 GCAAGATTTG CAAATCTTCC AGATATTTTA TTAAAAATGC GAACTGGTCG TAATCTTATG
8281 GCTCGACGTG GATTGTCATA TTTATTGTAC GAGTATAAAG TATTGTATTA TCAATATAAA
8341 ATTGGTTTTA TTCGAAAAAA TGAATTAATA AGTAATGCTA TGTTGAGAAC ATTTTTTCGT
8401 ATAATGCCAT CTAAATTAAA GGAGCTGATG TATTCAATCG TTAGGAATCG ATAATAATAA
8461 TTTTCTGATT AAGTGTTATG GATTTATTTT TATTAGGCAT ATTCTATAAT TAAGCATAAC
8521 CCGCATACCA CCCAGCGGTA TCCTGACAGG AGTAAACAAT GTCAAAGCAA CAGATCGGCG
8581 TCGTCGGTAT GGCAGTGATG GGGCGCAACC TTGCGCTCAA TATCGAAAGC CGTGGTTATA
8641 CCGTCTCTAT TTTCAACCGT TCCCGTGAAA AGACCGAAGA AGTGATTACC GAAAATCCAG
8701 GCAAGAAACT GGTTCCTTAC TATACGGTGA AAGAATTTGT TGAATCTCTG GAAACGCCTC
8761 GTCGCATCCT GTTAATGGTG AAAGCAGGTG CTGGCACGGA TGCTGCTATT GATTCCCTCA
8821 AGCCATACCT CGATAAAGGT GACATCATCA TTGATGGTGG TAACACCTTC TTCCATGACA
8881 CCATTCGTCG TAACCGTGAG CTTTCTGCAG AAGGCTTTAA CTTTATCGGT ACCGGTGTTT
8941 CCGGTGGTGA AGAAGGTGCG CTGAAAGGTC CTTCCATTAT GCCTGGTGGG CAGAAAGAAG
9001 CTTATGAACT GATTGCGCCG ATCCTGACCA AAATCGCCGC TGTGGCTGAA GACGGCGAAC
9061 CGTGCGTTAC CTATATTGGT GCCGATGGTG CAGGTCATTA TGTGAAGATG GTTCACAACG
9121 GTATTGAATA CGGTGATATG CAGCTGATTG CTGAAGCCTA TTCTCTGCTT AAAGGTGGCT
9181 TGAACCTCAC CAACGAAGAA CTGGCGCAGA CCTTTACCGA GTGGAATAAC GGTGAACTGA
9241 GCAGCTACCT GATCGACATC ACCAAAGATA TCTTCACCAA AAAAGATGAA GAGGGTAACT
9301 ACCTGGTTGA TGTGATTCTG GATGAAGCAG CAAACAAAGG TACGGGCAAA TGGACCAGCC
9361 AGAGCGCGCT GGATCTCGGC GAACCGCTGT CGCTGATTAC CGAGTCTGTG TTTGCACGTT
9421 ATATCTCTTC TCTGAAAGAG CAGCGTGTTG CCGCATCTAA AGTTCTCTCT GGCCCGCAAG
9481 CGCAGCCAGC TGGCGACAAT GCTGAGTTCA TCGAAAAAGT TCGCCGTGCG CTGTATCTGG
9541 GCAAAATCGT TTCTTACGCT CAGGGCTTCT CTCAGCTACG CGCTGCGTCT GAAGAGTACA
9601 ACTGGGATCT GAACTACGGT GAAATCGCGA AGATTTTCCG TGCTGGCTCC ATCATCCGTG
9661 CGCAGTTCCT GCAGAAAATC ACCGATGCTT ATGCCGAAAA TCCGCAGATC GCTAACCTGT
9721 TGCTGGCTCC TTACTTCAAG CAAATTGCCG ATGACTACCA GCAGGCGCTG CGCGATGTCG
9781 TCGCTTACGC AGTACAGAAC GGTATCCCGG TGCCCTACCT CGCCGCTGCG GTTGCCTATT
9841 ACGACAGCTA CCGCGCCGCT GTTCTGCCTG CGAACCTGAT CCAGGCACAG CGTGACTATT
9901 TCGGTGCGCA TACTTATAAG CGCATTGATA AAGAAGGTGT GTTCCATACC GAATGGCTGG
9961 ATTAATCTGA TTTAAATCAA TTAATCAAAG CAAGGCCCGG AGAAACCCTC CGGGCTTTTT
10021 TATTATACAA AGCGGCAGGT TAGGGCCTTT TTTTATAATT TATAGTTAAA AACGCGATAT
10081 AATACAGCGC CGCACAGCAG GATCGCTGCC TTGACAGTTC ATCTACATCA GCGTTAAAAA
10141 TCCCGCAGTA GATGAAGCTG TGGTGGTGGA TTAATGACCA CTCTAAATGT TTAACCGGAA
10201 GAAGTCAGAG CTAATGAAAA TAACAATTTC AGGAACAGGT TATGTTGGTC TTTCAAATGG
10261 TATTCTGATT GCGCAAAACC ACGAAGTGGT TGCACTGGAT ATCGTTCAGG CCAAAGTGGA
10321 CATGCTTAAC AAGAGGCAGT CACCGCTTGT TGATAAGGAG ATTGAAGAGT ATCTGGCGAC
10381 TAAAGATCTC AATTTCCGCG CTACGACAGA TAAGTATGAC GCGTATAAAA ATGCCGATTA
10441 CGTTATTATT GCCACACCTA CCGATTATGA TCCGAAAACA AATTACTTTA ATACCTCAAG
10501 CGTGGAAGCG GTCATTCGTG ATGTGACAGA AATTAATCCC AACGCGGTAA TGATTATAAA
10561 ATCAACTATC CCTGTTGGTT TTACAGAGTC CATTAAAGAA CGTTTTGGTA TTGAAAATGT
10621 GATCTTTTCG CCTGAGTTTT TGCGTGAAGG TAAAGCACTT TATGATAACT TACACCCATC
10681 ACGCATTGTG ATTGGCGAGC AGTCTGAACG CGCTAAACGT TTTGCTGCGT TATTACAGGA
10741 AGGCGCCATT AAGCAAGACA TACCAACATT GTTTACTGAC TCAACCGAGG CTGAGGCGAT
10801 TAAACTTTTT GCGAACACTT ATCTGGCGAT GCGTGTAGCG TATTTCAATG AACTTGATAG
10861 TTATGCTGAA AGCCTGGGAC TTAATTCACG CCAGATTATT GAGGGCGTAT GCCTTGACCC
10921 GCGTATCGGT AATCACTACA ACAACCCGTC ATTCGGTTAT GGTGGTTATT GTCTGCCGAA
10981 AGATACTAAG CAGTTACTGG CAAATTACCA GTCTGTGCCG AATAACCTGA TCTCGGCAAT
11041 TGTTGACGCC AACCGCACGC GCAAAGATTT TATTGCCGAT TCTATCCTTG CACGTAAACC
11101 GAAAGTTGTT GGCGTCTATC GTTTGATTAT GAAGAATGGT TCAGACAATT TTCGTGCTTC
11161 CTCGATTCAG GGTATTATGA AGCGAATCAA GGCGAAAGGT GTGCCTGTAA TCGTTTATGA
11221 GCCAGCTATG AAAGAGGACG ATTTTTTCCG GTCGCGCGTG GTACGTGATC TGGATGCGTT
11281 CAAACAAGAA GCTGATGTTA TTATTTCTAA CCGTATGTCT GCCGATCTGG CTGATGTAGC
11341 AGATAAAGTT TATACGCGCG ACTTGTTTGG CAATGATTAA TTATTTTGTT TCATTCTAAG
11401 AAAAGGCCCT AATAAATTAG GGCCTTTTCT TATGGTTTTG TAAAATCAAA CTTTATAGAA
11461 GTTACGATAC CATTCTACAA AGTTCTTTAC CCCTTCTTTA ACTGACGTTT CAGGTTTGAA
11521 TCCTATTACG TCATACAGTG CTTTTGTATC AGCACTGGTT TCCAGTACAT CACCGGGTTG
11581 GAGAGGCATC ATATTTTTGT TGGCTTCAAT ACCCAGAGCC TCTTCTAACG CATTGATATA
11641 GTCCATCAAC TCCACAGGCG AACTATTACC AATGTTATAG ACACGATATG GTGCTGAACT
11701 TGTTGCAGGC GAGCCTGTTT CTACAGCCCA CTGTGGGTTT TTTTCTGGAA TAACATCCTG
11761 TAAGCGAATA ATAGCTTCGG CAATATCATC AATGTAAGTA AAGTCACGCT TCATTTTGCC
11821 GAAGTTGTAA ACATCAATGC TTTTACCTTC CAGCATGGCT TTAGTGAATT TAAATAATGC
11881 CATATCCGGA CGTCCCCATG GACCATAAAC CGTAAAGAAA CGCAGCCCTG TGGTCGGTAA
11941 GCCATACAAA TGAGAATATG TATGGGCCAT GAGTTCATTC GCTTTTTTAG TTGCTGCATA
12001 AAGCGAAACA GGATGATCTA CAGAGTCATC TGTAGAGAAA GGCATCTTGC GGTTCATGCC
12061 ATAAACAGAA CTGGAGGAAG CGTAAAGTAG ATGCTGAACA TTATTATGGC GACATCCTTC
12121 TAGTATGTTC AGGAATCCAA TCAGGTTTGC ATCTGCATAT GCATTGGGAT TTTCAAGAGA
12181 GTAACGTACA CCGGCTTGCG CAGCGAGGTT TATTACGCGT TCGAACCGCT CGTCTGCAAA
12241 CAGTGCCGCC ATTTTCTCAC GATCGGCCAG GTCAATTTTA TAAAAACTGA AGTTGTCGTG
12301 CTTGAGTAAA TCAAGTCGTG CTTGTTTGAG GTTGACATCG TAATAATCAT TTAAGTTGTC
12361 AATGCCTACA ACCTGATGAC CAGCTGCAAG AAGCCGTTTA CTTAGATAGA AACCGATAAA
12421 GCCAGCAGCT CCCGTAACCA GAAATTTCAT TTATAATCCT CGCTCAGGCT AGAATATAGC
12481 CAATCTTCAT CTGGCATAAC TGAAAGTTAA ATTATACCGT TAGACAAGAA AAAAAGATAA
12541 TCGGTATCAG TTCTAAACTT GGCTGTTTTT TCTGGTAACG TGCTCATTTT ACAATCAAAG
12601 CTGTTCTAAG CTGACTATAC AAGCCGACGT CATTATCTCC AACCGTATGG CAGAAGAGCT
12661 TAAGGATGTG GCAGACAAAG TCTACACCCG CGATCTCTTT GGCAGTGACT AACATCCTGT
12721 TATCATGGCG ATTTTCGCCC TGATTCTCTT ATGTTCCCTT TGTAATAATT CATTATTTTT
12781 ATCATTTATC CTATAGCATT CATGGCGATT ATCGCTAAAC TATGGCGGCG CGCCACGTGG
12841 GATCCCCGGG TACCGAGCTC GAATTCGCCC TATAGTGAGT CGTATTACAA TTCACTGGCC
12901 GTCGTTTTAC AACGTCGTGA CTGGGAAAAC CCTGGCGTTA CCCAACTTAA TCGCCTTGCA
12961 GCACATCCCC CTTTCGCCAG CTGGCGTAAT AGCGAAGAGG CCCGCACCGA TCGCCCTTCC
13021 CAACAGTTGC GCAGCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC
13081 TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT
13141 AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GAACCCCTTG CGGCCGCATC
13201 GAATATAACT TCGTATAATG TATGCTATAC GAAGTTATTA GCGATGAGCT CGGACTTCCA
13261 TTGTTCATTC CACGGACAAA AACAGAGAAA GGAAACGACA GAGGCCAAAA AGCTCGCTTT
13321 CAGCACCTGT CGTTTCCTTT CTTTTCAGAG GGTATTTTAA ATAAAAACAT TAAGTTATGA
13381 CGAAGAAGAA CGGAAACGCC TTAAACCGGA AAATTTTCAT AAATAGCGAA AACCCGCGAG
13441 GTCGCCGCCC CGTAACCTGT CGGATCACCG GAAAGGACCC GTAAAGTGAT AATGATTATC
13501 ATCTACATAT CACAACGTGC GTGGAGGCCA TCAAACCACG TCAAATAATC AATTATGACG
13561 CAGGTATCGT ATTAATTGAT CTGCATCAAC TTAACGTAAA AACAACTTCA GACAATACAA
13621 ATCAGCGACA CTGAATACGG GGCAACCTCA TGTCCGAGCT CGCGAGCTCG TCGACAGCGA
13681 CACACTTGCA TCGGATGCAG CCCGGTTAAC GTGCCGGCAC GGCCTGGGTA ACCAGGTATT
13741 TTGTCCACAT AACCGTGCGC AAAATGTTGT GGATAAGCAG GACACAGCAG CAATCCACAG
13801 CAGGCATACA ACCGCACACC GAGGTTACTC CGTTCTACAG GTTACGACGA CATGTCAATA
13861 CTTGCCCTTG ACAGGCATTG ATGGAATCGT AGTCTCACGC TGATAGTCTG ATCGACAATA
13921 CAAGTGGGAC CGTGGTCCCA GACCGATAAT CAGACCGACA ACACGAGTGG GATCGTGGTC
13981 CCAGACTAAT AATCAGACCG ACGATACGAG TGGGACCGTG GTCCCAGACT AATAATCAGA
14041 CCGACGATAC GAGTGGGACC GTGGTTCCAG ACTAATAATC AGACCGACGA TACGAGTGGG
14101 ACCGTGGTCC CAGACTAATA ATCAGACCGA CGATACGAGT GGGACCATGG TCCCAGACTA
14161 ATAATCAGAC CGACGATACG AGTGGGACCG TGGTCCCAGT CTGATTATCA GACCGACGAT
14221 ACGAGTGGGA CCGTGGTCCC AGACTAATAA TCAGACCGAC GATACGAGTG GGACCGTGGT
14281 CCCAGACTAA TAATCAGACC GACGATACGA GTGGGACCGT GGTCCCAGTC TGATTATCAG
14341 ACCGACGATA CAAGTGGAAC AGTGGGCCCA GAGAGAATAT TCAGGCCAGT TATGCTTTCT
14401 GGCCTGTAAC AAAGGACATT AAGTAAAGAC AGATAAACGT AGACTAAAAC GTGGTCGCAT
14461 CAGGGTGCTG CCTTTTCAAG TTCCTTAAGA ATGGCCTCAA TTTTCTCTAT ACACTCAGTT
14521 GGAACACGAG ACCTGTCCAG GTTAAGCACC ATTTTATCGC CCTTATACAA TACTGTCGCT
14581 CCAGGAGCAA ACTGATGTCG TGAGCTTAAA CTAGTTCTTG ATGCAGATGA CGTTTTAAGC
14641 ACAGAAGTTA AAAGAGTGAT AACTTCTTCA GCTTCAAATA TCACCCCAGC TTTTTTCTGC
14701 TCATGAAGGT TAGATGCCTG CTGCTTAAGT AATTCCTCTT TATCTGTAAA TTTTTTTTGA
14761 AGTGCATCAC CTGACCGGGC AGATAGTTCA CCGGGGTGAG AAAAAAGAGC AACAACTGAT
14821 TTAGGCAATT TGGCGGTGTT GATACAGCGG GTAATAATCT TACGTGAAAT ATTTTCCGCA
14881 TCAGCCAGCG CAGAAATATT TCCAGCAAAT TCATTCTGCA ATCGGCTTGC ATAACGCTGA
14941 CCACGTTCAT AAGCACTTGT TGGGCGATAA TCGTTACCCA ATCTGGATAA TGCAGCCATC
15001 TGCTCATCAT CCAGCTCGCC AACCAGAACA CGATAATCAC TTTCGGTAAG TGCAGCAGCT
15061 TTACGACGGC GACTCCCATC GGCAATTTCT ATGACACCAG ATACTCTTCG ACCGAACGCC
15121 GGTGTCTGTT GACCAGTCAG TAGAAAAGAA GGGATGAGAT CATCCAGTGC GTCCTCAGTA
15181 AGCAGCTCCT GGTCACGTTC ATTACCTGAC CATACCCGAG AGGTCTTCTC AACACTATCA
15241 CCCCGGAGCA CTTCAAGAGT AAACTTCACA TCCCGACCAC ATACAGGCAA AGTAATGGCA
15301 TTACCGCGAG CCATTACTCC TACGCGCGCA ATTAACGAAT CCACCATCGG GGCAGCTGGT
15361 GTCGATAACG AAGTATCTTC AACCGGTTGA GTATTGAGCG TATGTTTTGG AATAACAGGC
15421 GCACGCTTCA TTATCTAATC TCCCAGCGTG GTTTAATCAG ACGATCGAAA ATTTCATTGC
15481 AGACAGGTTC CCAAATAGAA AGAGCATTTC TCCAGGCACC AGTTGAAGAG CGTTGATCAA
15541 TGGCCTGTTC AAAAACAGTT CTCATCCGGA TCTGACCTTT ACCAACTTCA TCCGTTTCAC
15601 GTACAACATT TTTTAGAACC ATGCTTCCCC AGGCATCCCG AATTTGCTCC TCCATCCACG
15661 GGGACTGAGA GCCATTACTA TTGCTGTATT TGGTAAGCAA AATACGTACA TCAGGCTCGA
15721 ACCCTTTAAG ATCAACGTTC TTGAGCAGAT CACGAAGCAT ATCGAAAAAC TGCAGTGCGG
15781 AGGTGTAGTC AAACAACTCA GCAGGCGTGG GAACAATCAG CACATCAGCA GCACATACGA
15841 CATTAATCGT GCCGATACCC AGGTTAGGCG CGCTGTCAAT AACTATGACA TCATAGTCAT
15901 GAGCAACAGT TTCAATGGCC AGTCGGAGCA TCAGGTGTGG ATCGGTGGGC AGTTTACCTT
15961 CATCAAATTT GCCCATTAAC TCAGTTTCAA TACGGTGCAG AGCCAGACAG GAAGGAATAA
16021 TGTCAAGCCC CGGCCAGCAA GTGGGCTTTA TTGCATAAGT GACATCGTCC TTTTCCCCAA
16081 GATAGAAAGG CAGGAGAGTG TCTTCTGCAT GAATATGAAG ATCTGGTACC CATCCGTGAT
16141 ACATTGAGGC TGTTCCCTGG GGGTCGTTAC CTTCCACGAG CAAAACACGT AGCCCCTTCA
16201 GAGCCAGATC CTGAGCAAGA TGAACAGAAA CTGAGGTTTT GTAAACGCCA CCTTTATGGG
16261 CAGCAACCCC GATCACCGGT GGAAATACGT CTTCAGCACG TCGCAATCGC GTACCAAACA
16321 CATCACGCAT ATGATTAATT TGTTCAATTG TATAACCAAC ACGTTGCTCA ACCCGTCCTC
16381 GAATTTCCAT ATCCGGGTGC GGTAGTCGCC CTGCTTTCTC GGCATCTCTG ATAGCCTGAG
16441 AAGAAACCCC AACTAAATCC GCTGCTTCAC CTATTCTCCA GCGCCGGGTT ATTTTCCTCG
16501 CTTCCGGGCT GTCATCATTA AACTGTGCAA TGGCGATAGC CTTCGTCATT TCATGACCAG
16561 CGTTTATGCA CTGGTTAAGT GTTTCCATGA GTTTCATTCT GAACATCCTT TAATCATTGC
16621 TTTGCGTTTT TTTATTAAAT CTTGCAATTT ACTGCAAAGC AACAACAAAA TCGCAAAGTC
16681 ATCAAAAAAC CGCAAAGTTG TTTAAAATAA GAGCAACACT ACAAAAGGAG ATAAGAAGAG
16741 CACATACCTC AGTCACTTAT TATCACTAGC GCTCGCCGCA GCCGTGTAAC CGAGCATAGC
16801 GAGCGAACTG GCGAGGAAGC AAAGAAGAAC TGTTCTGTCA GATAGCTCTT ACGCTCAGCG
16861 CAAGAAGAAA TATCCACCGT GGGAAAAACT CCAGGTAGAG GTACACACGC GGATAGCCAA
16921 TTCAGAGTAA TAAACTGTGA TAATCAACCC TCATCAATGA TGACGAACTA ACCCCCGATA
16981 TCAGGTCACA TGACGAAGGG AAAGAGAAGG AAATCAACTG TGACAAACTG CCCTCAAATT
17041 TGGCTTCCTT AAAAATTACA GTTCAAAAAG TATGAGAAAA TCCATGCAGG CTGAAGGAAA
17101 CAGCAAAACT GTGACAAATT ACCCTCAGTA GGTCAGAACA AATGTGACGA ACCACCCTCA
17161 AATCTGTGAC AGATAACCCT CAGACTATCC TGTCGTCATG GAAGTGATAT CGCGGAAGGA
17221 AAATACGATA TGAGTCGTCT GGCGGCCTTT CTTTTTCTCA ATGTATGAGA GGCGCATTGG
17281 AGTTCTGCTG TTGATCTCAT TAACACAGAC CTGCAGGAAG CGGCGGCGGA AGTCAGGCAT
17341 ACGCTGGTAA CTTTGAGGCA GCTGGTAACG CTCTATGATC CAGTCGATTT TCAGAGAGAC
17401 GATGCCTGAG CCATCCGGCT TACGATACTG ACACAGGGAT TCGTATAAAC GCATGGCATA
17461 CGGATTGGTG ATTTCTTTTG TTTCACTAAG CCGAAACTGC GTAAACCGGT TCTGTAACCC
17521 GATAAAGAAG GGAATGAGAT ATGGGTTGAT ATGTACACTG TAAAGCCCTC TGGATGGACT
17581 GTGCGCACGT TTGATAAACC AAGGAAAAGA TTCATAGCCT TTTTCATCGC CGGCATCCTC
17641 TTCAGGGCGA TAAAAAACCA CTTCCTTCCC CGCGAAACTC TTCAATGCCT GCCGTATATC
17701 CTTACTGGCT TCCGCAGAGG TCAATCCGAA TATTTCAGCA TATTTAGCAA CATGGATCTC
17761 GCAGATACCG TCATGTTCCT GTAGGGTGCC ATCAGATTTT CTGATCTGGT CAACGAACAG
17821 ATACAGCATA CGTTTTTGAT CCCGGGAGAG ACTATATGCC GCCTCAGTGA GGTCGTTTGA
17881 CTGGACGATT CGCGGGCTAT TTTTACGTTT CTTGTGATTG ATAACCGCTG TTTCCGCCAT
17941 GACAGATCCA TGTGAAGTGT GACAAGTTTT TAGATTGTCA CACTAAATAA AAAAGAGTCA
18001 ATAAGCAGGG ATAACTTTGT GAAAAAACAG CTTCTTCTGA GGGCAATTTG TCACAGGGTT
18061 AAGGGCAATT TGTCACAGAC AGGACTGTCA TTTGAGGGTG ATTTGTCACA CTGAAAGGGC
18121 AATTTGTCAC AACACCTTCT CTAGAACCAG CATGGATAAA GGCCTACAAG GCGCTCTAAA
18181 AAAGAAGATC TAAAAACTAT AAAAAAAATA ATTATAAAAA TATCCCCGTG GATAAGTGGA
18241 TAACCCCAAG GGAAGTTTTT TCAGGCATCG TGTGTAAGCA GAATATATAA GTGCTGTTCC
18301 CTGGTGCTTC CTCGCTCACT CGACCGGGAG GGTTCGAGAA GGGGGGGCAC CCCCCTTCGG
18361 CGTGCGCGGT CACGCGCACA GGGCGCAGCC CTGGTTAAAA ACAAGGTTTA TAAATATTGG
18421 TTTAAAAGCA GGTTAAAAGA CAGGTTAGCG GTGGCCGAAA AACGGGCGGA AACCCTTGCA
18481 AATGCTGGAT TTTCTGCCTG TGGACAGCCC CTCAAATGTC AATAGGTGCG CCCCTCATCT
18541 GTCAGCACTC TGCCCCTCAA GTGTCAAGGA TCGCGCCCCT CATCTGTCAG TAGTCGCGCC
13601 CCTCAAGTGT CAATACCGCA GGGCACTTAT CCCCAGGCTT GTCCACATCA TCTGTGGGAA
18661 ACTCGCGTAA AATCAGGCGT TTTCGCCGAT TTGCGAGGCT GGCCAGCTCC ACGTCGCCGG
18721 CCGAAATCGA GCCTGCCCCT CATCTGTCAA CGCCGCGCCG GGTGAGTCGG CCCCTCAAGT
18781 GTCAACGTCC GCCCCTCATC TGTCAGTGAG GGCCAAGTTT TCCGCGAGGT ATCCACAACG
18841 CCGGCGGCCG GCCGCGGTGT CTCGCACACG GCTTCGACGG CGTTTCTGGC GCGTTTGCAG
18901 GGCCATAGAC GGCCGCCAGC CCAGCGGCGA GGGCAACCAG CCGAGGGCTT CGCCCTGTCG
18961 CTCGACTGCG GCGAGCACTA CTGGCTGTAA AAGGACAGAC CACATCATGG TTCTGTGTTC
19021 ATTAGGTTGT TCTGTCCATT GCTGACATAA TCCGCTCCAC TTCAACGTAA CACCGCACGA
19081 AGATTTCTAT TGTTCCTGAA GGCATATTCA AATCGTTTTC GTTACCGCTT GCAGGCATCA
19141 TGACAGAACA CTACTTCCTA TAAACGCTAC ACAGGCTCCT GAGATTAATA ATGCGGATCT
19201 CTACGATAAT GGGAGATTTT CCCGACTGTT TCGTTCGCTT CTCAGTGGAT AACAGCCAGC
19261 TTCTCTGTTT AACAGACAAA AACAGCATAT CCACTCAGTT CCACATTTCC ATATAAAGGC
19321 CAAGGCATTT ATTCTCAGGA TAATTGTTTC AGCATCGCAA CCGCATCAGA CTCCGGCATC
19381 GCAAACTGCA CCCGGTGCCG GGCAGCCACA TCCAGCGCAA AAACCTTCGT GTAGACTTCC
19441 GTTGAACTGA TGGACTTATG TCCCATCAGG CTTTGCAGAA CTATCAGCGG TATACCGGCA
19501 TACAGCATGT GCATCGCATA GGAATGGCGG AACGTATGTG GTGTGACCGG AACAGAGAAC
19561 GTCACACCGT CAGCAGCAGC GGCGGCAACC GCCTCCCCAA TCCAGGTCCT GACCGTTCTG
19621 TCCGTCACTT CCCAGATCCG CGCTTTCTCT GTCCTTCCTG TGCGACGGTT ACGCCGCTCC
19681 ATGAGCTTAT CGCGAATAAA TACCTGTGAC GGAAGATCAC TTCGCAGAAT AAATAAATCC
19741 TGGTGTCCCT GTTGATACCG GGAAGCCCTG GGCCAACTTT TGGCGAAAAT GAGACGTTGA
19801 TCGGCACGTA AGAGGTTCCA ACTTTCACCA TAATGAAATA AGATCACTAC CGGGCGTATT
19861 TTTTGAGTTA TCGAGATTTT CAGGAGCTAA GGAAGCTAAA ATGGAGAAAA AAATCACTGG
19921 ATATACCACC GTTGATATAT CCCAATGGCA TCGTAACTAA CATTTTGAGG CATTTCAGTC
19981 AGTTGCTCAA TGTACCTATA ACCAGACCGT TCAGCTGGAT ATTACGGCCT TTTTAAAGAC
20041 CGTAAAGAAA AATAAGCACA AGTTTTATCC GGCCTTTATT CACATTCTTG CCCGCCTGAT
20101 GAATGCTCAT CCGGAATTTC GTATGGCAAT GAAAGACGGT GAGCTGGTGA TATGGGATAG
20161 TGTTCACCCT TGTTACACCG TTTTCCATGA GCAAACTGAA ACGTTTTCAT CGCTCTGGAG
20221 TGAATACCAC GACGATTTCC GGCAGTTTCT ACACATATAT TCGCAAGATG TGGCGTGTTA
20281 CGGTGAAAAC CTGGCCTATT TCCCTAAAGG GTTTATTGAG AATATGTTTT TCGTCTCAGC
20341 CAATCCCTGG GTGAGTTTCA CCAGTTTTGA TTTAAACGTG GCCAATATGG ACAACTTCTT
20401 CGCCCCCGTT TTCACCATGG GCAAATATTA TACGCAAGGC GACAAGGTGC TGATGCCGCT
20461 GGCGATTCAG GTTCATCATG CCCTTTGTGA TGGCTTCCAT GTCGGCAGAA TGCTTAATGA
20521 ATTACAACAG TACTGCGATG AGTGGCAGGG CGGGGCGTAA TTTTTTTAAG GCAGTTATTG
20581 GTGCCCTTAA ACGCCTGGTT GCTACGCCTG AATAAGTGAT AATAAGCGGA TGAATGGCAG
20641 AAATTCGATG ATAAGCTGTC AAACATGAGA ATTGGTCGAC GGCCCGGGCG GCCGCAAGGG
20701 GTTCGCGTTG GCCGATTCAT TAATGCAGCT GGCACGACAG GTTTCCCGAC TGGAAAGCGG
20761 GCAGTGAGCG CAACGCAATT AATGTGAGTT AGCTCACTCA TTAGGCACCC CAGGCTTTAC
20821 ACTTTATGCT TCCGGCTCGT ATGTTGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG
20881 GAAACAGCTA TGACCATGAT TACGCCAAGC TATTTAGGTG AGACTATAGA ATACTCAAGC
20941 TTGCATGCCT GCAGGTCGAC TCTAGAGGAT CCCACGACGT CG
Nucleotide Sequence for pCC1FOS cut (pFOS)
and S. flexneri 6 O-antigen with Z3206
Locus pFOS cut and O-antigen cut (Z3206+)
Definition Ligation of inverted S. flexneri 6 O antigen cluster
amplified with Z3206Nhe and wzzAscI cut with NheI and AscI into
pCC1FOS with MCS cassette cut with NheI and AscI
Features Location/Qualifiers
CDS complement(370..396)
/label=wzz′
CDS 748..1752
/label=uge
CDS complement(1818..3011)
/label=ugd
CDS complement(3233..4639)
/label=gnd
CDS complement(4744..5577)
/label=wfbZ
CDS complement(5574..6443)
/label=wfbY
CDS complement(6460..7647)
/label=wzy
CDS complement(7703..8935)
/label=wzx
CDS complement(8932..9489)
/label=rmlC
CDS complement(9494..10372)
/label=rmlA
CDS complement(10430..11329)
/label=rmlD
CDS complement(11329..12414)
/label=rmlB
CDS complement(12787..13680)
/label=galF
CDS complement(13912..14907)
/label=Z3206
CDS complement(15065..15097)
/label=′weaM
CDS complement(15525..16184)
/label=cat
CDS 16403..16750
/label=redF
CDS 18145..18900
/label=repE
CDS 19479..20654
/label=parA
CDS 20654..21625
/label=parB
Length: 22887 bp
Type: DNA circular UNA
Sequence:
SEQ ID NO: 29
1 GCGGCCGCAA GGGGTTCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG
61 CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT
121 GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT CAGCTGCGCA ACTGTTGGGA
181 AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC
241 AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC
301 CAGTGAATTG TAATACGACT CACTATAGGG CGAATTCGAG CTCGGTACCC GGGGATCCCA
361 CGTGGCGCGC CGCCATAGTT TAGCGATAAT CGCCATGAAT GCTATAGGAT AAATGATAAA
421 AATAATGAAT TATTACAAAG GGAACATAAG AGAATCAGGG CGAAAATCGC CATGATAACA
481 GGATGTTAGT CACTGCCAAA GAGATCGCGG GTGTAGACTT TGTCTGCCAC ATCCTTAAGC
541 TCTTCTGCCA TACGGTTGGA GATAATGACG TCGGCTTGTA TAGTCAGCTT AGAACAGCTT
601 TGATTGTAAA ATGAGCACGT TACCAGAAAA AACAGCCAAG TTTAGAACTG ATACCGATTA
661 TCTTTTTTTC TTGTCTAACG GTATAATTTA ACTTTCAGTT ATGCCAGATG AAGATTGGCT
721 ATATTCTAGC CTGAGCGAGG ATTATAAATG AAATTTCTGG TTACGGGAGC TGCTGGCTTT
781 ATCGGTTTCT ATCTAAGTAA ACGGCTTCTT GCAGCTGGTC ATCAGGTTGT AGGCATTGAC
841 AACTTAAATG ATTATTACGA TGTCAACCTC AAACAAGCAC GACTTGATTT ACTCAAGCAC
901 GACAACTTCA GTTTTTATAA AATTGACCTG GCCGATCGTG AGAAAATGGC GGCACTGTTT
961 GCAGACGAGC GGTTCGAACG CGTAATAAAC CTCGCTGCGC AAGCCGGTGT ACGTTACTCT
1021 CTTGAAAATC CCAATGCATA TGCAGATGCA AACCTGATTG GATTCCTGAA CATACTAGAA
1081 GGATGTCGCC ATAATAATGT TCAGCATCTA CTTTACGCTT CCTCCAGTTC TGTTTATGGC
1141 ATGAACCGCA AGATGCCTTT CTCTACAGAT GACTCTGTAG ATCATCCTGT TTCGCTTTAT
1201 GCAGCAACTA AAAAAGCGAA TGAACTCATG GCCCATACAT ATTCTCATTT GTATGGCTTA
1261 CCGACCACAG GGCTGCGTTT CTTTACGGTT TATGGTCCAT GGGGACGTCC GGATATGGCA
1321 TTATTTAAAT TCACTAAAGC CATGCTGGAA GGTAAAAGCA TTGATGTTTA CAACTTCGGC
1381 AAAATGAAGC GTGACTTTAC TTACATTGAT GATATTGCCG AAGCTATTAT TCGCTTACAG
1441 GATGTTATTC CAGAAAAAAA CCCACAGTGG GCTGTAGAAA CAGGCTCGCC TGCAACAAGT
1501 TCAGCACCAT ATCGTGTCTA TAACATTGGT AATAGTTCGC CTGTGGAGTT GATGGACTAT
1561 ATCAATGCGT TAGAAGAGGC TCTGGGTATT GAAGCCAACA AAAATATGAT GCCTCTCCAA
1621 CCCGGTGATG TACTGGAAAC CAGTGCTGAT ACAAAAGCAC TGTATGACGT AATAGGATTC
1681 AAACCTGAAA CGTCAGTTAA AGAAGGGGTA AAGAACTTTG TAGAATGGTA TCGTAACTTC
1741 TATAAAGTTT GATTTTACAA AACCATAAGA AAAGGCCCTA ATTTATTAGG GCCTTTTCTT
1801 AGAATGAAAC AAAATAATTA ATCATTGCCA AACAAGTCGC GCGTATAAAC TTTATCTGCT
1861 ACATCAGCCA GATCGGCAGA CATACGGTTA GAAATAATAA CATCAGCTTC TTGTTTGAAC
1921 GCATCCAGAT CACGTACCAC GCGCGACCGG AAAAAATCGT CCTCTTTCAT AGCTGGCTCA
1981 TAAACGATTA CAGGCACACC TTTCGCCTTG ATTCGCTTCA TAATACCCTG AATCGAGGAA
2041 GCACGAAAAT TGTCTGAACC ATTCTTCATA ATCAAACGAT AGACGCCAAC AACTTTCGGT
2101 TTACGTGCAA GGATAGAATC GGCAATAAAA TCTTTGCGCG TGCGGTTGGC GTCAACAATT
2161 GCCGAGATCA GGTTATTCGG CACAGACTGG TAATTTGCCA GTAACTGCTT AGTATCTTTC
2221 GGCAGACAAT AACCACCATA ACCGAATGAC GGGTTGTTGT AGTGATTACC GATACGCGGG
2281 TCAAGGCATA CGCCCTCAAT AATCTGGCGT GAATTAAGTC CCAGGCTTTC AGCATAACTA
2341 TCAAGTTCAT TGAAATACGC TACACGCATC GCCAGATAAG TGTTCGCAAA AAGTTTAATC
2401 GCCTCAGCCT CGGTTGAGTC AGTAAACAAT GTTGGTATGT CTTGCTTAAT GGCGCCTTCC
2461 TGTAATAACG CAGCAAAACG TTTAGCGCGT TCAGACTGCT CGCCAATCAC AATGCGTGAT
2521 GGGTGTAAGT TATCATAAAG TGCTTTACCT TCACGCAAAA ACTCAGGCGA AAAGATCACA
2581 TTTTCAATAC CAAAACGTTC TTTAATGGAC TCTGTAAAAC CAACAGGGAT AGTTGATTTT
2641 ATAATCATTA CCGCGTTGGG ATTAATTTCT GTCACATCAC GAATGACCGC TTCCACGCTT
2701 GAGGTATTAA AATAATTTGT TTTCGGATCA TAATCGGTAG GTGTGGCAAT AATAACGTAA
2761 TCGGCATTTT TATACGCGTC ATACTTATCT GTCGTAGCGC GGAAATTGAG ATCTTTAGTC
2821 GCCAGATACT CTTCAATCTC CTTATCAACA AGCGGTGACT GCCTCTTGTT AAGCATGTCC
2881 ACTTTGGCCT GAACGATATC CAGTGCAACC ACTTCGTGGT TTTGCGCAAT CAGAATACCA
2941 TTTGAAAGAC CAACATAACC TGTTCCTGAA ATTGTTATTT TCATTAGCTC TGACTTCTTC
3001 CGGTTAAACA TTTAGAGTGG TCATTAATCC ACCACCACAG CTTCATCTAC TGCGGGATTT
3061 TTAACGCTGA TGTAGATGAA CTGTCAAGGC AGCGATCCTG CTGTGCGGCG CTGTATTATA
3121 TCGCGTTTTT AACTATAAAT TATAAAAAAA GGCCCTAACC TGCCGCTTTG TATAATAAAA
3181 AAGCCCGGAG GGTTTCTCCG GGCCTTGCTT TGATTAATTG ATTTAAATCA GATTAATCCA
3241 GCCATTCGGT ATGGAACACA CCTTCTTTAT CAATGCGCTT ATAAGTATGC GCACCGAAAT
3301 AGTCACGCTG TGCCTGGATC AGGTTCGCAG GCAGAACAGC GGCGCGGTAG CTGTCGTAAT
3361 AGGCAACCGC AGCGGCGAAG GTCGGCACCG GGATACCGTT CTGTACTGCG TAAGCGACGA
3421 CATCGCGCAG CGCCTGCTGG TAGTCATCGG CAATTTGCTT GAAGTAAGGA GCCAGCAACA
3481 GGTTAGCGAT CTGCGGATTT TCGGCATAAG CATCGGTGAT TTTCTGCAGG AACTGCGCAC
3541 GGATGATGCA GCCAGCACGG AAAATCTTCG CGATTTCACC GTAGTTCAGA TCCCAGTTGT
3601 ACTCTTCAGA CGCAGCGCGT AGCTGAGAGA AGCCCTGAGC GTAAGAAACG ATTTTGCCCA
3661 GATACAGCGC ACGGCGAACT TTTTCGATGA ACTCAGCATT GTCGCCAGCT GGCTGCGCTT
3721 GCGGGCCAGA GAGAACTTTA GATGCGGCAA CACGCTGCTC TTTCAGAGAA GAGATATAAC
3781 GTGCAAACAC AGACTCGGTA ATCAGCGACA GCGGTTCGCC GAGATCCAGC GCGCTCTGGC
3841 TGGTCCATTT GCCCGTACCT TTGTTTGCTG CTTCATCCAG AATCACATCA ACCAGGTAGT
3901 TACCCTCTTC ATCTTTTTTG GTGAAGATAT CTTTGGTGAT GTCGATCAGG TAGCTGCTCA
3961 GTTCACCGTT ATTCCACTCG GTAAAGGTCT GCGCCAGTTC TTCGTTGGTG AGGTTCAAGC
4021 CACCTTTAAG CAGAGAATAG GCTTCAGCAA TCAGCTGCAT ATCACCGTAT TCAATACCGT
4081 TGTGAACCAT CTTCACATAA TGACCTGCAC CATCGGCACC AATATAGGTA ACGCACGGTT
4141 CGCCGTCTTC AGCCACAGCG GCGATTTTGG TCAGGATCGG CGCAATCAGT TCATAAGCTT
4201 CTTTCTGCCC ACCAGGCATA ATGGAAGGAC CTTTCAGCGC ACCTTCTTCA CCACCGGAAA
4261 CACCGGTACC GATAAAGTTA AAGCCTTCTG CAGAAAGCTC ACGGTTACGA CGAATGGTGT
4321 CATGGAAGAA GGTGTTACCA CCATCAATGA TGATGTCACC TTTATCGAGG TATGGCTTGA
4381 GGGAATCAAT AGCAGCATCC GTGCCAGCAC CTGCTTTCAC CATTAACAGG ATGCGACGAG
4441 GCGTTTCCAG AGATTCAACA AATTCTTTCA CCGTATAGTA AGGAACCAGT TTCTTGCCTG
4501 GATTTTCGGT AATCACTTCT TCGGTCTTTT CACGGGAACG GTTGAAAATA GAGACGGTAT
4561 AACCACGGCT TTCGATATTG AGCGCAAGGT TGCGCCCCAT CACTGCCATA CCGACGACGC
4621 CGATCTGTTG CTTTGACATT GTTTACTCCT GTCAGGATAC CGCTGGGTGG TATGCGGGTT
4681 ATGCTTAATT ATAGAATATG CCTAATAAAA ATAAATCCAT AACACTTAAT CAGAAAATTA
4741 TTATTATCGA TTCCTAACGA TTGAATACAT CAGCTCCTTT AATTTAGATG GCATTATACG
4801 AAAAAATGTT CTCAACATAG CATTACTTAT TAATTCATTT TTTCGAATAA AACCAATTTT
4861 ATATTGATAA TACAATACTT TATACTCGTA CAATAAATAT GACAATCCAC GTCGAGCCAT
4921 AAGATTACGA CCAGTTCGCA TTTTTAATAA AATATCTGGA AGATTTGCAA ATCTTGCATT
4981 ATGTACAATT AATAGGCTCC ACAATGCAAA ATCTTGAGAT TTTCTGAATG GAGGATAACC
5041 ACCAACAGCT AATACTGTAT TCTTTCTAAA AATTACAGAA GGATGGCTAA CTGCGCTTCG
5101 TTTCCTCGCG AATTTAACTA TTTCTCTATG TTCGAGAGGC ACTTTGCGTG TTGAAATAAA
5161 CTCCTCAGTA ACAGTTTCAA TTTCATCAAT AAAACTGCCA CATACATCTA TTTCTGAATT
5221 ATTAATCATA AAAGAAATTT GTTTCTCAAA CCGATGAGGC AAAGAAATAT CATCAGCATC
5281 CATTCTTGCC ACTAACTCAT TCCTACAAGC CTTTAATCCT TCATTTAAGG CATTAGCCAA
5341 TCCAACATTT CTAGGTAAAG GTACAAATGT TACTATTTTA TTGCCAACAT CATCAATGAA
5401 TGAATTTATA ATATCGATGT GTGTTTGATG GAGTTCTCCA TCTGCAACAA TTACTATTTG
5461 ATCTGGCTTA AGTGTTTGAT CGTGAAAAAT AGAGCGTAGA GCCACCTCAA AAAATTGCGG
5521 TAGATCATTT TTATAAATGC TAATTAAAAC TGAGAATTTT TCTAATCTAT GATTCATTTC
5581 ATTTTACCAC TTCGACCCAT TAAACCGTCA TTAATGCCTT TTAAAAAAAA ATATAACCTT
5641 TTATTACCAT TTGGAAGGAA AATAGGATAT AAAAAAACCT TTCCAATTAA TTTAACCAGA
5701 CTAGAAATTT TCCAGTAGAT GGGTACATAA TTTTTATTTA ATAAAAGAAA GATATTTCGA
5761 GTAGCATAAT AATGACGAAA TGGGCTTGGC AAACCGACAG AAAGAATATT TAAGATCTTA
5821 AATCGCCCAT CTCCAAGTCT ATGTGCAAGT AACGCATTTT TATTCCTAAT TACTTTAAAC
5881 CCAGCAGCTC TTAATCTCCA ACAATATTCA TGGTCTACCG CATCGATAAA AAGCTCATCT
5941 TTCATTCCTC CAACAATCAA CCAACTATTT TTTGGTATTA GACTGCCAGA ACTTAATGTA
6001 CTATCTACCT CATAATAAAC TTCTGTAAGT GGTTTCCCTT TTTTTACCCT TGCTTTATTT
6061 AATTCACCAG TTACTTTATC AAAATCTTGT GAACCAACTA AACCAACATT GACATTTTGT
6121 TTAAGCAATT TTTTGTAACA AGTAAGTAAC TGCTCTACCA TCTTAGGATC AGGAATACTA
6181 TCCTGATCCA TTTGCAATAT AAAATCAGCG CCATTTTCAA AAGCCCATTT CATTCCTATA
6241 CTTTGGGCTT CTGCTATGCC TAAATTATCA TTGAAATTGA ATATTTTTAC ATCGCCTGAA
6301 GAATTTTCAG CATATTTATA ACCATTTGTA GAGTTATTGC AAACGACAAC TTTAGTAACT
6361 TGTCTCAACA ATAATTCAAC CGCATTTTTT AAATCATTAT GTTCTGGGTT GTAAGCAACC
6421 AAAACGGCAT ATACAGTGTC CATCTTCACC TTAAAACCTT CATTTAGCTT TCATCTTTTT
6481 TAGAACATTA CTTAATGTCA CTAATACAAT TATTACAGCA ACATGGTTAG AGTCTAAAAT
6541 ATAAGGATTA GTAATTGCAT AAGAAACATA TAGAAAATAT AGCACACACA ACTCACTGTA
6601 TTTTATGATT TTAATCGTGA GAAGGAGATT AATTAATAAA AACAAAGTAA ATAAAATAAC
6661 GCCAAGTTGA TTTAAAAAAT AAACTGACTG CAATTCATAA TATATATATG CACTATAATC
6721 ACGGATAGGA GTTTGAATTT TGATGACATT ACCCAAACCA GAACCTATAA CAAAATTTGA
6781 TACAGACTCT GTAAGATCAT TAATTAATAC AGTAAACTGA TCCCATCTAA CTCCTAAAGA
6841 AGAATCAGCT CCATTTGATT TCATGATTAT CAACTCAATT GAATATGTAA TAAAAAAAGG
6901 GAGAATCACA GTAAGAAAAA CCCCAAAAAT AATTTTCCTT AATTTAGCGT ATCGTGAGTT
6961 AGATTTAGAA CATAGTATAA TATACATAAA AAACAAGCAT ATCGAAACAA AATATGCAAA
7021 ATTACCAGCC ACTATAGTAC CTATAGCCAG AATAACGGTT ATTGTATTTT TGAATCGATA
7081 ATAGAAATAA TCTTTTATGA CTATATGCAA CATAAAGGCA AATGGAATGA GAGCATTTCC
7141 TTTAATTTGA ACTCTATAGA AACCACTTCC ATATGTATAA ACATCACCAT AATCATTCTC
7201 CAAAAAATAA TGTCTTAGTG CTGAATAATC ACCAATACCA TATGTTTTTG TCATATAAAT
7261 ACTAATGATG GATATAATAA CCGCCTGTAA TACCATTAAA TATAAAAATA TTTTAACAAT
7321 CGAGATGGTT CCATAAGAGC AGAAATAAGC ACATAATATA AATAATATGA TAATATAAAA
7381 CCTAATTATT ATCGCTATAT CGTTACCCTT GATATAGGAA TAAATAAAAT TTATAAAAAG
7441 AGCTAATAGA AATATTAAAA TAACAGGATA GTGATATATT CCGTTTGCAA TTTTCTTTGT
7501 AAATGACATG ATACAAAGAC ATAAAAACCC CTCCATAATC CAACTATATT GAATAAATGG
7561 AAAGCTACGT GTAAGGAAAA ATATAAACCC AAAAAACAAA AGAACACTTA AACTTTTGTC
7621 TTTTGAGTTA TAAAAATCAG AAGTCATGTT TGCACTCTAA TTAGATGGGC TTGAGGAAGT
7681 AATCCCTAAA ATCAATTCGC TATTAATATT TCGTATCAAT TAATAATAAT ATCAAAAAAT
7741 CTAACGATGT TCTTACAGAC CATGCTATTG CGGCTCCAAC AATTCCCCAA TGATAAATAA
7801 AAATATATAA TATGCATAAA TATGGGATAA CTTCGAGCAA ATGAATAATA GCTGTAATTT
7861 TTGATCTTCC ACTAGCCTGA ACTGAAACAA ATGGGATTTG TGCAATGCAA TTAAAAAAGA
7921 AACCTATTGC AAGAATTTTT AATACTATAC CTGGCGTCCC ATGATATGTA GGTCCCATCC
7981 AAGCGGACAT TATAAAATCT GATAAAATAA TTATCAACAT TACAATTGGA AGTATACCAA
8041 TAACCATTAT AAAATATGAT AATATTTTAG TTTGCTTTAC CGATTGCAAT TCTGAACTTA
8101 ATCTTGGAAA AATAGCTCTG GACAACGCAC TTGGTAATAT CGTTAAGCGT TGTATACCTT
8161 CAGACGGAGC AGTATAAAAA GAAACTTTAT CAGCCCCCAC AATGTGTGAA AGAATAAAAC
8221 GATCCATATA TGTCATAATA GGGCTAATAA TATTGCTAAC TGTTATCCAG CTTCCAAAGC
8281 CGATTAATCT TTTAACTGTT ACAATTTTTA CAGACAGCCC AGATGATATT ATTAGTTTTC
8341 GACTAAATAT AAAGGTCACT ATAAGTGATA AGACTCTTGC CATAACTAAA CCATATATAG
8401 CACTTAGTAA TCCTCCATGA AAAAAACAGA AAATCACTGG TAATCCAGCC ACAAAAGAGT
8461 TGTTAATTGA TTTTATTAAA TTTACTTTTC TGAACTTTTC CATCCCCTCA AAAATCCCCA
8521 ACCAGACTTG GTTTAACAAG TATAAGGGTA TGGTAGCTGA AATAATATAT ATTGCTTTGA
8581 CAGATTCTAC AACATGATTC GCGTTAATGT TTAATAATTT AACAATTACA TTGCTACTCA
8641 AAAATAGTAC ACTACCGCCA ATCAAGCCCA ATATAGTTAG AATTACCGTT GAAGTTGAAA
8701 TGATCGCTCT TAATTCTTTA TGAACATTTT TATATATTGA TACTTCTCTT ATAACAGCTC
8761 TGGTCAATCC AGCATCAAAA ATACTTGCAT ATCCAACTAA GGCAATAGCT AACGTAAAAA
8821 GGCCAAATTG CTCGGTCCCT AGAATTCTAG ACAGTATACC TAACGCAGGA ATTGCTATTA
8881 ATGATGGTAT AATATACCCA CTTATATTCC ATAAAGTATT CTTTACAATA CTCACAAAAA
8941 TAATTCCTTC ATGTTATGCA ATTCTTTAGC CCTTGCATCT TTAATCGATA AAATATAATT
9001 ATTATGTTCT ATCGTCGGCC ATTTTATGCT CAGAATAGGA TCATTCCATA CAATCCCTCT
9061 ATCACTATCA GGATGATAAT AGTTCGTCGT TTTATATAAA AATTCCGCAG TCTCGCTCAG
9121 CACCAAAAAA CCATGTGCAA ATCCCTCAGG GATCCACAAT TGCCGCTTAT TCTCAGCAGA
9181 TAAATTCACC CCAACCCATT TACCAAAGGT AGGCGACGAT TTACGAATAT CAACAGCTAC
9241 ATCAAAAACC TCACCAACAA CGCAACGTAC CAGTTTCCCT TGCGCATAAG GTTCTAACTG
9301 ATAATGCAGC CCGCGTAAAA CACCTTTACT AGACTTCGAA TGGTTATCCT GAACAAATTC
9361 AACCTTACGT CCTACAGCTT CTTCGAAAAC TTTCTGATTA AAGCTTTCCA TAAAGAAACC
9421 ACGCTCATCA CCAAAAACTT TCGGCTCGAA AATTAACACA TCAGGAATTT CTGTTTTAAT
9481 TACGTTCATT TTATTAATAA CCTTTAATCA TTTTCAGCAG ATACTGTCCA TAAGCATTTT
9541 TTTTCAGCGC CTCCGCTAAT GCTTTCACCT GTTCAGCATC AATAAACCCT TTACGGTAAG
9601 CAATTTCTTC TGGGCAGGAA ACCTTTAGTC CCTGGCGCTC TTCAATGGTG GCAATGAAGT
9661 TGCTTGCTTC AATAAGACTC TGATGTGTCC CCGTATCCAG CCATGCATAA CCACGCCCCA
9721 TCATGGCAAC GGATAAACGC CCCTGTTCCA TATAAATACG GTTAATATCG GTAATTTCCA
9781 GTTCACCACG GGCAGAAGGC TTAAGGTTTT TCGCCATTTC GACAACGTCG TTATCATAGA
9841 AATAAAGCCC GGTTACCGCA TAATTACTTT TTGGTTGTAG CGGTTTTTCT TCCAGGCTTA
9901 TTGCCGTACC GTTTTTATCA AACTCAACGA CGCCGTAGCG TTCAGGATCA TTAACGTGAT
9961 AGGCAAATAC CGTTGCACCA CTTTCTTTGT TAACAGCGAC ATCCATTAAC TTCGGCAGAT
10021 CATGACCGTA GAAGATATTA TCACCAAGAA CCAAAGCACA ATCATCACCA CCGATAAACT
10081 CTTCACCGAT AATAAACGCC TGCGCAAGCC CATCTGGAGT CGGTTGCACT TTGTACTGAA
10141 GATTTAGCCC CCACTGGCTA CCGTCACCTA GCAGTTGTTG AAAACGAGGA GTATCCTGTG
10201 GCGTACTAAT AATCAGAATA TCGCGAATAC CCGCCAACAT CAGTGTAGAG AGCGGGTAAT
10261 AGATCATCGG CTTATCATAA ATAGGTAATA GCTGTTTACT GACAGCCATA GTCACAGGAT
10321 AAAGACGTGT ACCAGAACCA CCCGCTAAAA TAATACCTTT ACGCGTTTTC ATTTCATCAT
10381 TCCTTTTAAT TCATCTTGCT CCACCATCAC GAACAAGATG CAAAAACTAT TAAATTGCTG
10441 TAGTCGTAAT TAATTCGTTG AGCATTCGTT TCACACCAAC CTGCCAGTCA GGCAAGACAA
10501 GCGCAAAGTT CTGCTGAAAT TTTTCTGTAT TAAGGCGAGA GTTATGTGGA CGACGAGCTG
10561 GTGTAGGATA GGCTGTTGTT GGTACTGCGT TGAGCTTGTT GAGTGCAAGG GGAATACCTG
10621 CTTTGCGCGC CTCTTCAAAA ACCAGCGCAG CATAATCGTG CCAGGTTGTG GTACCACTGG
10681 CTACCAGATG GTACAAACCT GCGACTTCCG GTTTATTCAG TGCCACACGA ATAGCATGTG
10741 CCGTACAATC AGCCAGCAGC TCAGCACCTG TTGGCGCACC AAATTGATCA TTTATCACAG
10801 CCAGTTCTTC GCGCTCTTTT GCCAGACGCA ACATCGTTTT GGCGAAGTTA TTTCCTTTAG
10861 CTGCGTATAC CCAGCTGGTA CGGAAAATAA GATGCTTCGC GCAATGTTCC TGTAACGCTT
10921 TTTCTCCGGC TAACTTGGTT TCACCGTAAA CATTTAGCGG TGCGGTTGCA TCCGTCTCCA
10981 GCCATGGCGT GTCGCCATTT CCAGGGAATA CGTAGTCAGT TGAGTAATGA ATTACCCAAG
11041 CCCCAACTTC ATTAGCCTCT TTTGCAATTG ATTCAACACT AGTCGCATTG AGTAATTGTG
11101 CAAATTCGGG TTCTGACTCA GCCTTATCTA CTGCGGTGTG AGCCGCAGCA TTAACAATAA
11161 CATCAGGTCG AATTCTTTTG ACTGTTTCAG CTACACCTTC AGGATTACTA AAATCACCAC
11221 AATAATCAGT GGAGTGAACA TCAAGAGCAA TCAAATTACC CAAAGGTGCC AGAGCACGCT
11281 GTAGTTCCCA ACCTACCTGC CCTGTTTTGC CGAAAAGGAG GATATTCATT ACTGGCGGCC
11341 CTCATAGTTC TGTTCAATCC ACGATTGATA AGCACCACTT TTCACATTAT CAACCCATTT
11401 TGTATTGGAC AGGTACCATT CCAATGTCTT CCGAATCCCG CTCTCAAACG TTTCCTGCGG
11461 TTTCCAGCCC AATTCGCGGC TAATCTTCTC TGCATCAATC GCATAACGGC GATCGTGTCC
11521 CGGGCGATCG GCAACATAAG TAATTTGCTC GCGGTAAGAT TTCTCTTTCG GTACAATCTC
11581 ATCCAGCAAA TCACAAATAG TGAGCACTAC ATCGATGTTT TTCTTTTCGT TGTGTCCACC
11641 AATGTTATAA GTTTCACCCG CTTTACCTTC GGTTACGACG GTATATAACG CACGCGCATG
11701 ATCTTCAACA TACAGCCAGT CACGAATTTG ATCCCCTTTG CCATAAATAG GTAATGCCTT
11761 ACCTTCCAGA GCATTCAGAA TAACCAATGG AATCAATTTT TCCGGGAAAT GATAAGGACC
11821 ATAATTATTA GAGCAATTAG TCACAATGGT TGGTAAACCA TAGGTACGTT TCCACGCGCG
11881 GACTAAATGA TCGCTGGATG CTTTTGAAGC GGAATAAGGG CTGCTTGGCG CGTAAGCTGT
11941 TGTCTCTGTA AATAAGGGTA ATTCTTCTGT ATTATTTACC TCGTCAGGAT GAGGCAAATC
12001 ACCATAGACT TCGTCAGTAG AAATATGATG AAAACGGAAT CTAGTTTTCT TGTCGCTATC
12061 AAGAGCAGAC CAATAATTGC GAGCGGCTTC CAAAAGGACA TATGTACCAA CAATATTGGT
12121 TTCAATAAAT GCCGCAGGAC CTGTAATTGA ACGGTCAACA TGGCTTTCAG CAGCCAGGTG
12181 CATCACTGCA TCTGGCTGAT GCTGAGCAAA AATCCGTGCC ATTGCAGCTG CATCGCAAAT
12241 ATCCGCATGT TCAAAAACAT AGCGTTCAGA ATCAGAAACA TCAGCAAGTG ATTCCAGGTT
12301 TCCGGCGTAC GTTAATTTAT CGACATTAAC AACACTATCC TGCGTATTAT TTATAATGTG
12361 ACGAACTACA GCAAAACCAA TAAATCCTGC GCCACCAGTA ACAAGTATTT TCACCTAATT
12421 TATTCCATAT TGCTTCAGAG CATGCTGTGA AATAAGCGGC TCTCAGTTTG ATTAATAGAA
12481 GTATTAATGC ACGCTACCGC CCCTGGCTTT ACAGCTACCA GAGCACTGCA TGCATGCCTA
12541 CGATGTGACG AGCGTTACCC ACTCGCGCTA AACCCGAAAA ATTCAAAAGC TAATTGTCTT
12601 ACCAATCCGC TCTGGAAACA AGGAAAATCC TGGAAAACTT TGACTAAAAT CCTATTGCTA
12661 ACTCGTTGTT ATTCTGATTG TTTATATAAA ACAACGGCAG GAATATTCGC AACAAATTAC
12721 TTTCACCACG AATCTTCACT GCCGTTATAA TTTTCTTATC AACCGTTACA TCCGGTCAGA
12781 TTTTCATTAT TCGCTTAACA GCTTCTCAAT ACCTTTACGG AACTTCGCCC CTTCTTTCAG
12841 GTTGCGCAGC CCATACTTCA CAAACGCCTG CATATAGCCC ATTTTTTTAC CGCAGTCGTA
12901 GCTGTCGCCG GTCATCAGCA TTGCATCAAC GGACTGTTTT TTCGCCAGCT CGGCAATGGC
12961 ATCAGTCAGC TGAATACGTC CCCATGCACC AGGCTGAGTA CGTTCAAGTT CCGGCCAAAT
13021 ATCGGCAGAA AGCACATAGC GACCAACGGC CATGATGTCT GAGTCCAGCG TCTGCGGCTG
13081 ATCCGGTTTT TCGATAAATT CAACAATGCG GCTGACTTTA CCTTCGCGAT CCAGCGGTTC
13141 TTTGGTCTGG ATGACGGAGT ATTCAGAGAG GTCACCCGGC ATACGTTTTG CCAGCACCTG
13201 GCTACGGCCC GTTTCATTGA AGCGCGCAAT CATGGCAGCA AGGTTGTAGC GTAGCGGGTC
13261 GGCGCTGGCG TCGTCGATCA CAACGTCTGG CAGCACCACG ACAAATGGAT TGTCACCAAT
13321 GGCGGGTCGT GCACACAAAA TGGAGTGACC TAAACCTAAA GGTTCGCCCT GACGCACGTT
13381 CATAATAGTC ACGCCCGGCG GGCAGATAGA TTGCACTTCC GCCAGTAGTT GACGCTTCAC
13441 GCGCTGCTCA AGGAGAGATT CTAATTCATA AGAGGTGTCG AAGTGGTTTT CGACCGCGTT
13501 CTTGGACGCA TGAGTTACCA GGAGGATTTC TTTGATCCCT GCAGCCACAA TCTCGTCAAC
13561 AATGTACTGA ATCATTGGCT TGTCGACGAT CGGTAGCATC TCTTTGGGTA TCGCCTTAGT
13621 GGCAGGCAAC ATATGCATCC CAAGACCCGC TACCGGTATA ACTGCTTTTA AATTCGTCAT
13681 TATTTTCCTA CCTCTAAGGG GCTGATAGTG CGTAAATTAT TGTCATAGGT TAGCCAAACG
13741 GTATGGCTAT ATACCAAGCA TAACTTTGAT TAAACCTTAC GATAACACTA CACACCATCA
13801 GCATCTGGGT TACTCGGATT ACTCGGAAAT CCACATACTG ATAATTTAAT CAGTACCTCT
13861 TTCCGAATAA TCGTAGTCCA ACCTGGTCCT TTTTTCTCTG ACTCGTCTGC ATTACTCAGA
13921 AACAAACGTT ATGTCGTCTT TTTTGGCATG GACGAATTCA TACTGCAGAG TTCGATCCAG
13981 ACCTTGCGAC AGCGTATACG GTGCAACAAA ACCTGAAGAA TGCACTTTCG TTGCGTCAAA
14041 CTGTGTTGTT GCGCAGAATT TTTTCACGCG CACAGAGCTG ACAGCGTATT TTTTGCCCGT
14101 AATTTTGCTC AGGATATCAA AGCAATATCC ACCCAGCATT CCTAGTGGGT AAGGCAAGTG
14161 CATAGAAGGG ATCTTTTTGT TCAGGCTTTG TTCAACTTCA GCAACCAACT GGTTCATGTT
14221 CAGGTCTGGC TTATCAACAT AGTTATAAAC CTCATAACCT GCGGCAACAT TCTTCAGTTT
14281 GTACTTGATA AACTCAACAA TGTTTCCAAC ATAAGCCATG GACTTATAGT TAGTCCCTGC
14341 GCCCACCATC ATAAACTTGC CGCCAGCGAT CTGTTTCAGC AAGTTATAGA CGTTACCGCG
14401 GTTGCGTTCA CCGAAGATAA CGGTAGGACG GATGATGGTT AATGAACGTT CTGTTGGTGC
14461 TTTGTTATAC CATTCACGCA GCACTTCCTC TGCCTGCCAC TTACTTTTGC CGTAGTGGTT
14521 GAAAGGGTCG TGTGGATGGT TTTCGTCAGG GTTGTGTTTG TTCAAACCAT AAACAGCAAC
14581 GGAACTGGTA AAGATGATAT TTTTAACGCC ATTTTTTTCC ATGGCCGCCA GCACATTGCG
14641 GGTACCCTGA ACGTTGACAT CATAATAGAG AGAAGTAGGG CTGACGTCAT CGCGGTGTTC
14701 CGCTGCCAGT AGTACAACAG TGTCAAAACC GGCTAACGCC TGGTCGAGTG CCTGTTGATC
14761 ACGAACATCA CCAATCTGTG TGATTTCTGG ATAAAAGTGG CTCTGCCGTT TGTCCAGGTT
14821 CTTGATATTA AAGTCAGCAA TTGCCGTTTC AAGTAGTCGG GTTCCTACGA ATCCGGAAGC
14881 TCCTATGAGC AAAACGTTAT TGTTCATAAA TCACTTTAGT CTGGTTGTTA CGTAAGAAAC
14941 ACAAGATAAA GATGAGTACC TTCCCTGAGT AGTCAATGCT GCCCAGCCCC AGCTTTAACA
15001 GTTAGTGTGA GGATTATAAT CTTTTAGAAC ATTATATCCA GTAAGTTTAT GAATGGTCGC
15061 AAATCTACTC TCTCCGTTCC GGCAATCTAA AGTTAATGCT AGCGACGTCG TGGGATCCTC
15121 TAGAGTCGAC CTGCAGGCAT GCAAGCTTGA GTATTCTATA GTCTCACCTA AATAGCTTGG
15181 CGTAATCATG GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA
15241 ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA
15301 CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC
15361 ATTAATGAAT CGGCCAACGC GAACCCCTTG CGGCCGCCCG GGCCGTCGAC CAATTCTCAT
15421 GTTTGACAGC TTATCATCGA ATTTCTGCCA TTCATCCGCT TATTATCACT TATTCAGGCG
15481 TAGCAACCAG GCGTTTAAGG GCACCAATAA CTGCCTTAAA AAAATTACGC CCCGCCCTGC
15541 CACTCATCGC AGTACTGTTG TAATTCATTA AGCATTCTGC CGACATGGAA GCCATCACAA
15601 ACGGCATGAT GAACCTGAAT CGCCAGCGGC ATCAGCACCT TGTCGCCTTG CGTATAATAT
15661 TTGCCCATGG TGAAAACGGG GGCGAAGAAG TTGTCCATAT TGGCCACGTT TAAATCAAAA
15721 CTGGTGAAAC TCACCCAGGG ATTGGCTGAG ACGAAAAACA TATTCTCAAT AAACCCTTTA
15781 GGGAAATAGG CCAGGTTTTC ACCGTAACAC GCCACATCTT GCGAATATAT GTGTAGAAAC
15841 TGCCGGAAAT CGTCGTGGTA TTCACTCCAG AGCGATGAAA ACGTTTCAGT TTGCTCATGG
15901 AAAACGGTGT AACAAGGGTG AACACTATCC CATATCACCA GCTCACCGTC TTTCATTGCC
15961 ATACGAAATT CCGGATGAGC ATTCATCAGG CGGGCAAGAA TGTGAATAAA GGCCGGATAA
16021 AACTTGTGCT TATTTTTCTT TACGGTCTTT AAAAAGGCCG TAATATCCAG CTGAACGGTC
16081 TGGTTATAGG TACATTGAGC AACTGACTGA AATGCCTCAA AATGTTCTTT ACGATGCCAT
16141 TGGGATATAT CAACGGTGGT ATATCCAGTG ATTTTTTTCT CCATTTTAGC TTCCTTAGCT
16201 CCTGAAAATC TCGATAACTC AAAAAATACG CCCGGTAGTG ATCTTATTTC ATTATGGTGA
16261 AAGTTGGAAC CTCTTACGTG CCGATCAACG TCTCATTTTC GCCAAAAGTT GGCCCAGGGC
16321 TTCCCGGTAT CAACAGGGAC ACCAGGATTT ATTTATTCTG CGAAGTGATC TTCCGTCACA
16381 GGTATTTATT CGCGATAAGC TCATGGAGCG GCGTAACCGT CGCACAGGAA GGACAGAGAA
16441 AGCGCGGATC TGGGAAGTGA CGGACAGAAC GGTCAGGACC TGGATTGGGG AGGCGGTTGC
16501 CGCCGCTGCT GCTGACGGTG TGACGTTCTC TGTTCCGGTC ACACCACATA CGTTCCGCCA
16561 TTCCTATGCG ATGCACATGC TGTATGCCGG TATACCGCTG AAAGTTCTGC AAAGCCTGAT
16621 GGGACATAAG TCCATCAGTT CAACGGAAGT CTACACGAAG GTTTTTGCGC TGGATGTGGC
16681 TGCCCGGCAC CGGGTGCAGT TTGCGATGCC GGAGTCTGAT GCGGTTGCGA TGCTGAAACA
16741 ATTATCCTGA GAATAAATGC CTTGGCCTTT ATATGGAAAT GTGGAACTGA GTGGATATGC
16801 TGTTTTTGTC TGTTAAACAG AGAAGCTGGC TGTTATCCAC TGAGAAGCGA ACGAAACAGT
16861 CGGGAAAATC TCCCATTATC GTAGAGATCC GCATTATTAA TCTCAGGAGC CTGTGTAGCG
16921 TTTATAGGAA GTAGTGTTCT GTCATGATGC CTGCAAGCGG TAACGAAAAC GATTTGAATA
16981 TGCCTTCAGG AACAATAGAA ATCTTCGTGC GGTGTTACGT TGAAGTGGAG CGGATTATGT
17041 CAGCAATGGA CAGAACAACC TAATGAACAC AGAACCATGA TGTGGTCTGT CCTTTTACAG
17101 CCAGTAGTGC TCGCCGCAGT CGAGCGACAG GGCGAAGCCC TCGGCTGGTT GCCCTCGCCG
17161 CTGGGCTGGC GGCCGTCTAT GGCCCTGCAA ACGCGCCAGA AACGCCGTCG AAGCCGTGTG
17221 CGAGACACCG CGGCCGGCCG CCGGCGTTGT GGATACCTCG CGGAAAACTT GGCCCTCACT
17281 GACAGATGAG GGGCGGACGT TGACACTTGA GGGGCCGACT CACCCGGCGC GGCGTTGACA
17341 GATGAGGGGC AGGCTCGATT TCGGCCGGCG ACGTGGAGCT GGCCAGCCTC GCAAATCGGC
17401 GAAAACGCCT GATTTTACGC GAGTTTCCCA CAGATGATGT GGACAAGCCT GGGGATAAGT
17461 GCCCTGCGGT ATTGACACTT GAGGGGCGCG ACTACTGACA GATGAGGGGC GCGATCCTTG
17521 ACACTTGAGG GGCAGAGTGC TGACAGATGA GGGGCGCACC TATTGACATT TGAGGGGCTG
17581 TCCACAGGCA GAAAATCCAG CATTTGCAAG GGTTTCCGCC CGTTTTTCGG CCACCGCTAA
17641 CCTGTCTTTT AACCTGCTTT TAAACCAATA TTTATAAACC TTGTTTTTAA CCAGGGCTGC
17701 GCCCTGTGCG CGTGACCGCG CACGCCGAAG GGGGGTGCCC CCCCTTCTCG AACCCTCCCG
17761 GTCGAGTGAG CGAGGAAGCA CCAGGGAACA GCACTTATAT ATTCTGCTTA CACACGATGC
17821 CTGAAAAAAC TTCCCTTOGG GTTATCCACT TATCCACGGG GATATTTTTA TAATTATTTT
17881 TTTTATAGTT TTTAGATCTT CTTTTTTAGA GCGCCTTGTA GGCCTTTATC CATGCTGGTT
17941 CTAGAGAAGG TGTTGTGACA AATTGCCCTT TCAGTGTGAC AAATCACCCT CAAATGACAG
18001 TCCTGTCTGT GACAAATTGC CCTTAACCCT GTGACAAATT GCCCTCAGAA GAAGCTGTTT
18061 TTTCACAAAG TTATCCCTGC TTATTGACTC TTTTTTATTT AGTGTGACAA TCTAAAAACT
18121 TGTCACACTT CACATGGATC TGTCATGGCG GAAACAGCGG TTATCAATCA CAAGAAACGT
18181 AAAAATAGCC CGCGAATCGT CCAGTCAAAC GACCTCACTG AGGCGGCATA TAGTCTCTCC
18241 CGGGATCAAA AACGTATGCT GTATCTGTTC GTTGACCAGA TCAGAAAATC TGATGGCACC
18301 CTACAGGAAC ATGACGGTAT CTGCGAGATC CATGTTGCTA AATATGCTGA AATATTCGGA
18361 TTGACCTCTG CGGAAGCCAG TAAGGATATA CGGCAGGCAT TGAAGAGTTT CGCGGGGAAG
18421 GAAGTGGTTT TTTATCGCCC TGAAGAGGAT GCCGGCGATG AAAAAGGCTA TGAATCTTTT
18481 CCTTGGTTTA TCAAACGTGC GCACAGTCCA TCCAGAGGGC TTTACAGTGT ACATATCAAC
18541 CCATATCTCA TTCCCTTCTT TATCGGGTTA CAGAACCGGT TTACGCAGTT TCGGCTTAGT
18601 GAAACAAAAG AAATCACCAA TCCGTATGCC ATGCGTTTAT ACGAATCCCT GTGTCAGTAT
18661 CGTAAGCCGG ATGGCTCAGG CATCGTCTCT CTGAAAATCG ACTGGATCAT AGAGCGTTAC
18721 CAGCTGCCTC AAAGTTACCA GCGTATGCCT GACTTCCGCC GCCGCTTCCT GCAGGTCTGT
18781 GTTAATGAGA TCAACAGCAG AACTCCAATG CGCCTCTCAT ACATTGAGAA AAAGAAAGGC
18841 CGCCAGACGA CTCATATCGT ATTTTCCTTC CGCGATATCA CTTCCATGAC GACAGGATAG
18901 TCTGAGGGTT ATCTGTCACA GATTTGAGGG TGGTTCGTCA CATTTGTTCT GACCTACTGA
18961 GGGTAATTTG TCACAGTTTT GCTGTTTCCT TCAGCCTGCA TGGATTTTCT CATACTTTTT
19021 GAACTGTAAT TTTTAAGGAA GCCAAATTTG AGGGCAGTTT GTCACAGTTG ATTTCCTTCT
19081 CTTTCCCTTC GTCATGTGAC CTGATATCGG GGGTTAGTTC GTCATCATTG ATGAGGGTTG
19141 ATTATCACAG TTTATTACTC TGAATTGGCT ATCCGCGTGT GTACCTCTAC CTGGAGTTTT
19201 TCCCACGGTG GATATTTCTT CTTGCGCTGA GCGTAAGAGC TATCTGACAG AACAGTTCTT
19261 CTTTGCTTCC TCGCCAGTTC GCTCGCTATG CTCGGTTACA CGGCTGCGGC GAGCGCTAGT
19321 GATAATAAGT GACTGAGGTA TGTGCTCTTC TTATCTCCTT TTGTAGTGTT GCTCTTATTT
19381 TAAACAACTT TGCGGTTTTT TGATGACTTT GCGATTTTGT TGTTGCTTTG CAGTAAATTG
19441 CAAGATTTAA TAAAAAAACG CAAAGCAATG ATTAAAGGAT GTTCAGAATG AAACTCATGG
19501 AAACACTTAA CCAGTGCATA AACGCTGGTC ATGAAATGAC GAAGGCTATC GCCATTGCAC
19561 AGTTTAATGA TGACAGCCCG GAAGCGAGGA AAATAACCCG GCGCTGGAGA ATAGGTGAAG
19621 CAGCGGATTT AGTTGGGGTT TCTTCTCAGG CTATCAGAGA TGCCGAGAAA GCAGGGCGAC
19681 TACCGCACCC GGATATGGAA ATTCGAGGAC GGGTTGAGCA ACGTGTTGGT TATACAATTG
19741 AACAAATTAA TCATATGCGT GATGTGTTTG GTACGCGATT GCGACGTGCT GAAGACGTAT
19801 TTCCACCGGT GATCGGGGTT GCTGCCCATA AAGGTGGCGT TTACAAAACC TCAGTTTCTG
19861 TTCATCTTGC TCAGGATCTG GCTCTGAAGG GGCTACGTGT TTTGCTCGTG GAAGGTAACG
19921 ACCCCCAGGG AACAGCCTCA ATGTATCACG GATGGGTACC AGATCTTCAT ATTCATGCAG
19981 AAGACACTCT CCTGCCTTTC TATCTTGGGG AAAAGGACGA TGTCACTTAT GCAATAAAGC
20041 CCACTTGCTG GCCGGGGCTT GACATTATTC CTTCCTGTCT GGCTCTGCAC CGTATTGAAA
20101 CTGAGTTAAT GGGCAAATTT GATGAAGGTA AACTGCCCAC CGATCCACAC CTGATGCTCC
20161 GACTGGCCAT TGAAACTGTT GCTCATGACT ATGATGTCAT AGTTATTGAC AGCGCGCCTA
20221 ACCTGGGTAT CGGCACGATT AATGTCGTAT GTGCTGCTGA TGTGCTGATT GTTCCCACGC
20281 CTGCTGAGTT GTTTGACTAC ACCTCCGCAC TGCAGTTTTT CGATATGCTT CGTGATCTGC
20341 TCAAGAACGT TGATCTTAAA GGGTTCGAGC CTGATGTACG TATTTTGCTT ACCAAATACA
20401 GCAATAGTAA TGGCTCTCAG TCCCCGTGGA TGGAGGAGCA AATTCGGGAT GCCTGGGGAA
20461 GCATGGTTCT AAAAAATGTT GTACGTGAAA CGGATGAAGT TGGTAAAGGT CAGATCCGGA
20521 TGAGAACTGT TTTTGAACAG GCCATTGATC AACGCTCTTC AACTGGTGCC TGGAGAAATG
20581 CTCTTTCTAT TTGGGAACCT GTCTGCAATG AAATTTTCGA TCGTCTGATT AAACCACGCT
20641 GGGAGATTAG ATAATGAAGC GTGCGCCTGT TATTCCAAAA CATACGCTCA ATACTCAACC
20701 GGTTGAAGAT ACTTCGTTAT CGACACCAGC TGCCCCGATG GTGGATTCGT TAATTGCGCG
20761 CGTAGGAGTA ATGGCTCGCG GTAATGCCAT TACTTTGCCT GTATGTGGTC GGGATGTGAA
20821 GTTTACTCTT GAAGTGCTCC GGGGTGATAG TGTTGAGAAG ACCTCTCGGG TATGGTCAGG
20881 TAATGAACGT GACCAGGAGC TGCTTACTGA GGACGCACTG GATGATCTCA TCCCTTCTTT
20941 TCTACTGACT GGTCAACAGA CACCGGCGTT CGGTCGAAGA GTATCTGGTG TCATAGAAAT
21001 TGCCGATGGG AGTCGCCGTC GTAAAGCTGC TGCACTTACC GAAAGTGATT ATCGTGTTCT
21061 GGTTGGCGAG CTGGATGATG AGCAGATGGC TGCATTATCC AGATTGGGTA ACGATTATCG
21121 CCCAACAAGT GCTTATGAAC GTGGTCAGCG TTATGCAAGC CGATTGCAGA ATGAATTTGC
21181 TGGAAATATT TCTGCGCTGG CTGATGCGGA AAATATTTCA CGTAAGATTA TTACCCGCTG
21241 TATCAACACC GCCAAATTGC CTAAATCAGT TGTTGCTCTT TTTTCTCACC CCGGTGAACT
21301 ATCTGCCCGG TCAGGTGATG CACTTCAAAA AGCCTTTACA GATAAAGAGG AATTACTTAA
21361 GCAGCAGGCA TCTAACCTTC ATGAGCAGAA AAAAGCTGGG GTGATATTTG AAGCTGAAGA
21421 AGTTATCACT CTTTTAACTT CTGTGCTTAA AACGTCATCT GCATCAAGAA CTAGTTTAAG
21481 CTCACGACAT CAGTTTGCTC CTGGAGCGAC AGTATTGTAT AAGGGCGATA AAATGGTGCT
21541 TAACCTGGAC AGGTCTCGTG TTCCAACTGA GTGTATAGAG AAAATTGAGG CCATTCTTAA
21601 GGAACTTGAA AAGCCAGCAC CCTGATGCGA CCACGTTTTA GTCTACGTTT ATCTGTCTTT
21661 ACTTAATGTC CTTTGTTACA GGCCAGAAAG CATAACTGGC CTGAATATTC TCTCTGGGCC
21721 CACTGTTCCA CTTGTATCGT CGGTCTGATA ATCAGACTGG GACCACGGTC CCACTCGTAT
21781 CGTCGGTCTG ATTATTAGTC TGGGACCACG GTCCCACTCG TATCGTCGGT CTGATTATTA
21841 GTCTGGGACC ACGGTCCCAC TCGTATCGTC GGTCTGATAA TCAGACTGGG ACCACGGTCC
21901 CACTCGTATC GTCGGTCTGA TTATTAGTCT GGGACCATGG TCCCACTCGT ATCGTCGGTC
21961 TGATTATTAG TCTGGGACCA CGGTCCCACT CGTATCGTCG GTCTGATTAT TAGTCTGGAA
22021 CCACGGTCCC ACTCGTATCG TCGGTCTGAT TATTAGTCTG GGACCACGGT CCCACTCGTA
22081 TCGTCGGTCT GATTATTAGT CTGGGACCAC GATCCCACTC GTGTTGTCGG TCTGATTATC
22141 GGTCTGGGAC CACGGTCCCA CTTGTATTGT CGATCAGACT ATCAGCGTGA GACTACGATT
22201 CCATCAATGC CTGTCAAGGG CAAGTATTGA CATGTCGTCG TAACCTGTAG AACGGAGTAA
22261 CCTCGGTGTG CGGTTGTATG CCTGCTGTGG ATTGCTGCTG TGTCCTGCTT ATCCACAACA
22321 TTTTGCGCAC GGTTATGTGG ACAAAATACC TGGTTACCCA GGCCGTGCCG GCACGTTAAC
22381 CGGGCTGCAT CCGATGCAAG TGTGTCGCTG TCGACGAGCT CGCGAGCTCG GACATGAGGT
22441 TGCCCCGTAT TCAGTGTCGC TGATTTGTAT TGTCTGAAGT TGTTTTTACG TTAAGTTGAT
22501 GCAGATCAAT TAATACGATA CCTGCGTCAT AATTGATTAT TTGACGTGGT TTGATGGCCT
22561 CCACGCACGT TGTGATATGT AGATGATAAT CATTATCACT TTACGGGTCC TTTCCGGTGA
22621 TCCGACAGGT TACGGGGCGG CGACCTCGCG GGTTTTCGCT ATTTATGAAA ATTTTCCGGT
22681 TTAAGGCGTT TCCGTTCTTC TTCGTCATAA CTTAATGTTT TTATTTAAAA TACCCTCTGA
22741 AAAGAAAGGA AACGACAGGT GCTGAAAGCG AGCTTTTTGG CCTCTGTCGT TTCCTTTCTC
22801 TGTTTTTGTC CGTGGAATGA ACAATGGAAG TCCGAGCTCA TCGCTAATAA CTTCGTATAG
22861 CATACATTAT ACGAAGTTAT ATTCGAT
